Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice.

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.

ADME pharmacogenetics: investigation of the pharmacokinetics of the antiretroviral agent lopinavir coformulated with ritonavir.

BACKGROUND: An ADME (absorption, distribution, metabolism and excretion)-pharmacogenetics association study may identify functional variants relevant to the pharmacokinetics of lopinavir co-formulated with ritonavir (LPV/r), a first-line anti-HIV agent. METHODS: An extensive search of literature and web resources helped select ADME genes and single nucleotide polymorphisms (SNPs, functional and HapMap tagging SNPs) with a proven or potentially relevant role in LPV/r pharmacokinetics. The study followed a two-stage design. Stage 1 (discovery) considered a Caucasian population (n=638) receiving LPV/r, where we selected 117 individuals with low LPV clearance (cases) and 90 individuals with high clearance (controls). Genotyping was performed by a 1536-SNP customized GoldenGate Illumina BeadArray. Stage 2 (confirmation) represented a replication study of candidate SNPs from the stage 1 in 148 individuals receiving LPV/r. The analysis led to formal population pharmacokinetic-pharmacogenetic modeling of demographic, environmental and candidate SNP effects. RESULTS: One thousand three hundred and eighty SNPs were successfully genotyped. Nine SNPs prioritized by the stage 1 analysis were brought to replication. Stage 2 confirmed the contribution of two functional SNPs in SLCO1B1, one functional SNP in ABCC2 and a tag SNP of the CYP3A locus in addition to body weight effect and ritonavir coadministration. According to the population pharmacokinetic-pharmacogenetic model, genetic variants explained 5% of LPV variability. Individuals homozygous rs11045819 (SLCO1B1*4) had a clearance of 12.6 l/h, compared with 5.4 l/h in the reference group, and 3.9 l/h in individuals with two or more variant alleles of rs4149056 (SLCO1B1*5), rs717620 (ABCC2) or rs6945984 (CYP3A). A subanalysis confirmed that although a significant part of the variance in LPV clearance was attributed to fluctuation in ritonavir levels, genetic variants had an additional effect on LPV clearance. CONCLUSION: The two-stage strategy successfully identified genetic variants affecting LPV/r pharmacokinetics. Such a general approach of ADME pharmacogenetics should be generalized to other drugs.

Role of CLOCK and circadian rhythms in calcium homeostasis

Le maintien d'une concentration sanguine constante de calcium est d'une importance cruciale et trois organes participent à la balance calcique normale : les reins, les intestins et les os. La concentration plasmatique de calcium est strictement régulée par l'hormone parathyroïdienne (PTH) et par la vitamine D. Des variations circadiennes de la PTH, de la vitamine D ainsi que du calcium plasmatique ont été décrites précédemment chez l'humain ainsi que chez le rat. Ces rythmes de PTH dans le sérum sont importants pour la régulation du remodelage de l'os. En effet, il a été montré chez les souris C57BL/6J que des injections de PTH une fois par jour mènent à une augmentation de la densité minérale de l'os alors que l'infusion en continu de PTH est associée à une diminution de cette densité. La vitamine D joue également un rôle fondamental dans la physiologie osseuse, car un déficit en vitamine D peut conduire à une ostéomalacie. Cependant la fonction des oscillations de vitamine D au niveau de l'homéostasie osseuse reste inconnue. L'horloge circadienne est un système interne de contrôle biologique du temps générant des rythmes de 24 heures dans l'expression des gènes, ainsi que dans la physiologie et le comportement. Ce contrôle s'opère par des boucles rétroactives positives et négatives de l'expression de gènes circadiens tels que CLOCK, BMAL1, CRY1 et 2 ou PERI et 2. Dans ce travail, nous avons émis l'hypothèse que l'homéostasie calcique est sous le contrôle de l'horloge circadienne. Dans un premier temps, nous avons montré chez les souris C57BL/6J des variations journalières des concentrations de calcium, de PTH et de vitamine D dans le sang, ainsi que de calcium dans les urines. Nous avons également démontré des changements au niveau de l'expression rénale des gènes importants dans l'homéostasie du calcium, tant au niveau de l'ARN messager que des protéines. Ensuite, pour analyser le rôle du système de l'horloge circadienne dans l'homéostasie du calcium, nous avons étudié des souris dans lesquelles a été supprimé le gène CLOCK crucial pour la fonction de l'horloge et nous avons comparé ces souris à des souris de type sauvage de même portée. Les souris CLOCK-I- étaient hypercalciuriques à chaque moment de la journée. Cependant le rythme circadien de l'excrétion de calcium était préservé. Le taux de calcium plasmatique ne différait pas entre les génotypes, mais les souris CLOCK -/- ne montraient pas de variations journalières de ce paramètre. Une perte du rythme journalier était également observée pour les niveaux de vitamine D, perte qui pourrait être une cause de l'altération de la micro-architecture osseuse révélée chez les souris CLOCK-/-. En effet, ces souris montrent une diminution du nombre de trabécules, de leur volume ainsi que de leur surface, ce qui suggère la présence d'ostéoporose. Nous avons également trouvé que le rythme de l'expression de l'ARN messager de CYP27B1 était aboli dans les reins des souris CLOCK -/-, ce qui peut expliquer l'altération du rythme de la vitamine D. Les taux sanguins de PTH étaient comparables entre les souris CLOCK -/- et de type sauvage. Dans les reins, une augmentation de l'expression de l'ARN messager de TRPV5 et NCX1 a été constatée, ce qui suggérerait une augmentation de la réabsorption de calcium dans le tubule convoluté distal et dans le tubule connecteur. Dans les intestins, la réabsorption calcique était diminuée, chez les souris CLOCK-I-, fait confirmé par une diminution des niveaux d'ARN messager de TRPV6 et PMCAL. En résumé, la suppression du gène CLOCK chez les souris a conduit à une hypercalciurie, une altération du rythme des taux plasmatiques de calcium et de vitamine D et à une détérioration de l'architecture osseuse. Pour conclure, ces résultats montrent que l'horloge circadienne est essentielle à l'homéostasie calcique ainsi qu'à la physiologie des os. - L'ostéoporose affecte environ 22 millions de femmes et 5.5 millions d'hommes en Europe, réduisant significativement leur qualité de vie et a causé 3.5 millions de nouvelles fractures en 2010. Les dépenses totales liées à ces fractures ont atteint 37 milliards d'euro et ce coût devrait augmenter de 25% d'ici à 2025. Le nombre de nouvelles fractures dues à l'ostéoporose à travers le monde est estimé à environ 1000 par heure. Parmi les causes de l'ostéoporose, le déficit én calcium et/ou en vitamine D joue un rôle important, mais il existe également des causes génétiques ou liées à des facteurs comme les hormones sexuelles (estrogènes, testostérone), l'âge, le tabac, le poids corporel, certains médicaments,... La vie est rythmique : ceci est dû à l'alternance naturelle du jour et de la nuit et de ses effets sur le corps. La prise alimentaire, par exemple, est un processus qui a lieu pendant la phase active, qui est prévisible (il se produit toujours au même moment) et qui peut être anticipé par le corps. Pour cela, une horloge interne est présente dans chaque cellule du corps et est synchronisée par la lumière du jour, entre autres stimuli. Cette horloge indique la phase du jour et régule l'expression de gènes impliqués dans les différents processus qui nécessitent une anticipation. Pendant mon travail de thèse, je me suis demandé si des îythmes circadiens (c'est-à-dire d'une durée d'environ 24 heures et indépendants des stimuli externes) étaient observables'pour les gènes régulant les flux de calcium dans le corps et si l'interruption de ces rythmes pouvait mener à des altérations de la qualité de l'os. J'ai d'abord travaillé avec des souris normales et j'ai pu montrer la présence de rythmes au niveau du calcium sanguin et urinaire, mais également au niveau des hormones et gènes qui contrôlent le métabolisme du calcium dans le corps, comme la vitamine D et l'hormone parathyroidienne. De manière intéressante, j'ai observé que la plupart de ces gènes ont un rythme synchronisé. J'ai ensuite utilisé un modèle de souris dans lequel l'horloge interne a été génétiquement invalidée et j'ai montré que ces souris présentent une augmentation de leur excrétion urinaire de calcium et un rythme circadien altéré de la vitamine D dans le sang. Ces souris absorbent aussi moins bien le calcium intestinal et présentent une ostéoporose marquée. Ce travail montre donc que l'horloge interne est nécessaire pour établir un rythme circadiens de certains facteurs influant les flux de calcium dans l'organisme, comme la vitamine D, et que la perturbation de ces rythmes mène à une dérégulation du métabolisme osseux. Ainsi, la perturbation de l'horloge interne peut causer une ostéoporose et une hypercalciurie qui pourraient aboutir à la formation de fractures et de calculs rénaux. L'extrapolation de ces observations chez l'homme ou à des changements plus subtiles des rythmes circadiens, comme le décalage horaire, restent à montrer. Cette recherche a démontré que les rythmes circadiens des mécanismes de régulation des flux de calcium dans l'organisme sont essentiels au maintien d'un squelette normal et suggère que les perturbations des rythmes circadiens pourraient être une nouvelle cause de l'ostéoporose. - Maintaining constant calcium concentration in the plasma is of a crucial importance and three organs participate in normal calcium balance - kidney, gut and bone. Plasma calcium concentration is strictly regulated by parathyroid hormone (PTH) and vitamin D. Circadian variations of PTH, vitamin D and plasma calcium were previously described in humans, as well as in rats. Rhythms in serum PTH are important for balanced bone remodelling. Indeed in C57BL/6J mice, PTH injection once per day leads to an increase in bone mineral density (BMD), whilst continuous infusion is associated with decreased BMD. Vitamin D also plays a crucial role in bone physiology, since the deficiency in vitamin D can lead to rickets/osteomalacia. However, the role of vitamin D rhythms in bone homeostasis remains unknown. The circadian clock is an. internal time-keeping system generating rhythms in gene expression with 24h periodicity, in physiology and in behaviour. It is operated by positive- and negative-feedback loops of circadian genes, such as CLOCK, BMAL1, CRY1 and 2 or PERI and 2. In this work, we hypothesized, that calcium homeostasis is under the control of the circadian clock. First, we showed daily variations in urinary calcium and serum calcium, PTH and l,25(OH)2 vitamin D, together with renal mRNA and protein levels of genes involved in calcium homeostasis in C57BL/6J mice. Second, and to investigate the role of the circadian clock system in calcium handling, we studied mice lacking the gene CLOCK crucial for fonction of the clock system and compared them to the WT littermates. CLOCK-/- mice were hypercalciuric at all timepoints of the day. However, the circadian rhythm of calcium excretion was preserved. Serum calcium levels did not differ between the genotypes, but CLOCK-/- mice did not exhibit daily variation for this parameter. Loss of rhythm was observed also for serum l,25(OH)2 vitamin D levels, which may be one of the causes of altered bone microarchitecture that was revealed in CLOCK-/- mice. They displayed increased trabecular separation and decreased trabecular number, trabecular bone volume and trabecular bone surface, suggestive of osteoporosis. We found that the rhythm of the mRNA expression of CYP27B1 was abolished in the kidney of CLOCK-/- mice, which could induce the altered rhythm of l,25(OH)2 vitamin. Serum PTH levels were comparable between CLOCK-/- and WT mice. In the kidney, increased mRNA expression of TRPV5 and NCX1 suggests increased calcium reabsorption in the distal convoluted and connecting tubule. In the gut, intestinal calcium absorption was decreased in CLOCK¬/- mice, confirmed by decreased mRNA levels of TRPV6 and PMCA1. In summary, deletion of the CLOCK gene in mice conducts to hypercalciuria, alteration of the rhythm in serum calcium and l,25(OH)2D levels, and impainnent of their bone microarchitecture. In conclusion, these data show that the circadian clock system is essential in calcium homeostasis and bone physiology.

Alkaline mineral water lowers bone resorption even in calcium sufficiency: alkaline mineral water and bone metabolism.

BACKGROUND: Dietary acid charge enhances bone loss. Bicarbonate or alkali diet decreases bone resorption in humans. We compared the effect of an alkaline mineral water, rich in bicarbonate, with that of an acid one, rich in calcium only, on bone markers, in young women with a normal calcium intake. METHODS: This study compared water A (per litre: 520 mg Ca, 291 mg HCO(3)(-), 1160 mg SO(4)(-), Potential Renal Acid load (PRAL) +9.2 mEq) with water B (per litre: 547 mg Ca, 2172 mg HCO(3)(-), 9 mg SO(4)(-), PRAL -11.2 mEq). 30 female dieticians aged 26.3 yrs (SD 7.3) were randomized into two groups, followed an identical weighed, balanced diet (965 mg Ca) and drank 1.5 l/d of the assigned water. Changes in blood and urine electrolytes, C-telopeptides (CTX), urinary pH and bicarbonate, and serum PTH were measured after 2 and 4 weeks. RESULTS: The two groups were not different at baseline, and showed a similar increase in urinary calcium excretion. Urinary pH and bicarbonate excretion increased with water B, but not with water A. PTH (p=0.022) and S-CTX (p=0.023) decreased with water B but not with water A. CONCLUSION: In calcium sufficiency, the acid calcium-rich water had no effect on bone resorption, while the alkaline water rich in bicarbonate led to a significant decrease of PTH and of S-CTX.

Whole body protein synthesis and energy expenditure in very low birth weight infants.

The aim of the present work was to study whole body protein synthesis and breakdown, as well as energy metabolism, in very low birth weight premature infants (less than 1500 g) during their rapid growth phase. Ten very low birth weight infants were studied during their first and second months of life. They received a mean energy intake of 114 kcal/kg X day and 3 g protein/kg X day as breast milk or milk formula. The average weight gain was 15 g/kg X day. The apparent energy digestibility was 88%, i.e. 99 kcal/kg X day. Their resting postprandial energy expenditure was 58 kcal/kg X day, indicating that 41 kcal/kg X day was retained. The apparent protein digestibility was 89%, i.e. 2.65 g/kg X day. Their rate of protein oxidation was 0.88 g/kg X day so that protein retention was 1.76 g/kg X day. There was a linear relationship between N retention and N intake (r = 0.78, p less than 0.001). The slope of the regression line indicates a net efficiency of N utilization of 67%. Estimates of body composition from the energy balance, coupled with N balance method, showed that 25% of the gain was fat and 75% was lean tissue. Whole body protein synthesis and breakdown were determined using repeated oral administration of 15N glycine for 60-72 h, and 15N enrichment in urinary urea was measured. Protein synthesis averaged 11.2 g/kg X day and protein breakdown 9.4 g/kg X day. Muscular protein breakdown, as estimated by 3-methylhistidine excretion, contributed to 12% of the total protein breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)

The epithelial sodium channel and the control of sodium balance.

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na(+) absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure. Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na(+) absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na(+) absorption in the ASDN, and in the amount of Na(+) that is excreted in the final urine. A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems. These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na(+) in response to Na(+) diet occurs predominantly in the early part (the connecting tubule) of the ASDN. An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na(+) absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na(+) balance.

Absorption and pharmacokinetics of green tea catechins in beagles

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12·35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 02, 26, 68 and 824 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for (2)-epigallocatechin-3-gallate (EGCG), (2)-epicatechin-3-gallate (ECG), (2)-epigallocatechin glucuronide (EGCglucuronide), (2)-epicatechin glucuronide (EC-glucuronide), (2)-epicatechin sulphate (EC sulphate) were 0·3 (range 0·11·9), 0·1 (range 00·4), 0·8 (range 0·23·9), 0·2 (range 0·1 1·7) and 1 (range 0·33·4) mmol/l, respectively. The areas under the plasma concentration v. time curves (AUC0!24) were 427 (range 1021185) mmol/l £ min for EGC-glucuronide, 112 (range 53919) mmol/l £ min for EC-sulphate, 71 (range 26306) mmol/l £ min for EGCG, 40 (range 12258) mmol/l £ min for EC-glucuronide and 14 (range 0·1124) mmol/l £ min for ECG. The values of mean residence time (MRT0!24) were 5 (range 216), 2 (range 111), 10 (range 213), 3 (range 216) and 2·4 (range 118) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

Four-hour infusions of synthetic atrial natriuretic peptide in normal volunteers.

Two doses of synthetic atrial natriuretic peptide (0.5 and 5.0 micrograms/min) and its vehicle were infused intravenously for 4 hours in eight salt-loaded normal volunteers, and the effect on blood pressure, heart rate, renal hemodynamics, solute excretion, and secretion of vasoactive hormones was studied. The 0.5 micrograms/min infusion did not alter blood pressure or heart rate, whereas the 5.0 micrograms/min infusion significantly reduced the mean pressure by 20/9 mm Hg after 2.5 to 3 hours and increased the heart rate slightly. Inulin clearance was not significantly changed, but the mean p-aminohippurate clearance fell by 13 and 32% with the lower and higher doses, respectively. Urinary excretion of sodium and chloride increased slightly with the lower dose. With the higher dose, a marked increase in urinary excretion of sodium, chloride, and calcium was observed, reaching a peak during the second hour of the infusion. Potassium and phosphate excretion did not change significantly. A brisk increase in urine flow rate and fractional water excretion was seen only during the first hour of the high-dose infusion. Signs and symptoms of hypotension were observed in two subjects. No change in plasma renin activity, angiotensin II, or aldosterone was observed during either infusion, but a marked increase occurred after discontinuation of the high-dose infusion. In conclusion, the 5 micrograms/min infusion induced a transient diuretic effect, delayed maximal natriuretic activity, and a late fall in blood pressure, with no change in inulin clearance but a dose-related decrease in p-aminohippurate clearance. Despite large amounts of sodium excreted and blood pressure reduction, no counterregulatory changes were observed in the renin-angiotensin-aldosterone system or plasma vasopressin levels during the infusion.

Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

Development of a hepatocyte hollow fiber bioreactor for the study of contrast agents by magnetic resonance imaging

Thanks to the continuous progress made in recent years, medical imaging has become an important tool in the diagnosis of various pathologies. In particular, magnetic resonance imaging (MRI) permits to obtain images with a remarkably high resolution without the use of ionizing radiation and is consequently widely applied for a broad range of conditions in all parts of the body. Contrast agents are used in MRI to improve tissue discrimination. Different categories of contrast agents are clinically available, the most widely used being gadolinium chelates. One can distinguish between extracellular gadolinium chelates such as Gd-DTPA, and hepatobiliary gadolinium chelates such as Gd-BOPTA. The latter are able to enter hepatocytes from where they are partially excreted into the bile to an extent dependent on the contrast agent and animal species. Due to this property, hepatobiliary contrast agents are particularly interesting for the MRI of the liver. Actually, a change in signal intensity can result from a change in transport functions signaling the presence of impaired hepatocytes, e.g. in the case of focal (like cancer) or diffuse (like cirrhosis) liver diseases. Although the excretion mechanism into the bile is well known, the uptake mechanisms of hepatobiliary contrast agents into hepatocytes are still not completely understood and several hypotheses have been proposed. As a good knowledge of these transport mechanisms is required to allow an efficient diagnosis by MRI of the functional state of the liver, more fundamental research is needed and an efficient MRI compatible in vitro model would be an asset. So far, most data concerning these transport mechanisms have been obtained by MRI with in vivo models or by a method of detection other than MRI with cellular or sub-cellular models. Actually, no in vitro model is currently available for the study and quantification of contrast agents by MRI notably because high cellular densities are needed to allow detection, and no metallic devices can be used inside the magnet room, which is incompatible with most tissue or cell cultures that require controlled temperature and oxygenation. The aim of this thesis is thus to develop an MRI compatible in vitro cellular model to study the transport of hepatobiliary contrast agents, in particular Gd-BOPTA, into hepatocytes directly by MRI. A better understanding of this transport and especially of its modification in case of hepatic disorder could permit in a second step to extrapolate this knowledge to humans and to use the kinetics of hepatobiliary contrast agents as a tool for the diagnosis of hepatic diseases.

Comparative efficacy of chelators to remove renal cadmium burden in isolated perfused rat kidneys

Trois agents chélateurs (l?acide diéthylène triamine penta-acétique, DTPA; l?acide méso-2,3- dimercaptosucchique, DMSA; l?acide 2,3-dimercapto- 1 -propanesulfonique, DMPS) ont été comparés quant à leur efficacité à mobiliser du cadmium (Cd) accumulé dans le tissu rénal. Des reins prélevés chez des rats exposés durant 3 j au Cd (acétate de Cd , 0.75 mg/kg.j, i.p) ont été isolés et perfusés in vitro, à l?aide d?un système de reperfusion utilisant une solution de Krebs-Henseleit, pH 7.4, contenant 8 acides aminés et 6% d?albumine. Les concentrations de Cd dans le perfusat et l?urine ont été mesurées par spectrométrie d?absorption atomique. Six périodes de clearance, après une période d?équilibration de 20 min, ont ?été obtenues. Le DMSA et le DMPS ont mobilisé le Cd à partir du tissu rénal, comme l?ont montré les augmentations dose-dépendantes des concentrations de Cd dans l?urine et le perfusat. L?accumulation de Cd était nettement plus élevée dans le perfusat que dans l?urine, indiquant que l?effet des chélateurs se marquait surtout au niveau tubulaire basolatéral. Le DTPA n?induisait qu?une faible mobilisation de Cd dans l?urine et le perfusat, et son efficacité était clairement inférieure à celle des autres chélateurs. Comme prévu, la quantité de Cd présente dans le tissu rénal après perfùsion par le DMSA ou le DMPS diminuait en fonction de l?efficacité des chélateurs, jusqu?à des valeurs inférieures de 46% au taux rénal de Cd avant perfusion. Le DMPS apparaissait induire une excretion urinaire de Cd plus importante que celle induite par le DMSA, une caractéristique qui pourrait être liée à une sécrétion tubulaire du chélateur, qui a été décrite antérieurement. Un intervalle de temps prolongé (1 -2 semaines) entre le moment de l?administration du Cd et la perfusion du rein avec le DMPS induisait une augmentation de l?excrétion urinaire de Cd. Tous les chélateurs se sont montrés néphrotoxiques à concentrations élevées.

Relación entre el nivel de grasa e ingestión y la excreción urinaria de nitrógeno y energía, en gazapos en crecimiento-cebo

Se determinó la excreción urinaria de nitrógeno (NU, %) y de energía (EU, kcal/100g), en la última semana de vida, de 36 conejos alimentados, desde el destete (a los 28 d) hasta el sacrificio a los dos meses de edad, con tres piensos conteniendo 0, 3 y 6 % de grasa añadida (G0, G3 y G6); y administrados a dos niveles de ingestión: ad libitum (IL) y restringido (IR) al 70 % de IL. El NU y la EU estuvieron influidos positivamente tanto por el nivel de ingestión (P<0,001) como por el % de grasa ingerida (P<0,05). En estos resultados puede estar involucrado el metabolismo tan elevado de las lipoproteínas transportadoras de lípidos, implicadas en el metabolismo de las grasas, ya sean de origen alimentario o de la lipogénesis de novo. La relación entre la EU y el NU aporta valores muy superiores a los que podría corresponder a las materias orgánicas nitrogenadas, por lo que se podría pensar, como posible causa, en la intervención de materia orgánica no nitrogenada. Asimismo, la cuantificación de la EM del pienso se ve afectada por el incremento del valor calórico de la orina, con el nivel de ingestión y/o el 6 % de grasa añadida. Con ello se evidencia la desigualdad entre ED y EM de la grasa en las raciones que las incluya. Asimismo, el hecho de que la movilización grasa —bien sea de origen alimentario o de la lipogénesis— altere la excreción nitrogenada y/o energética a través de la orina, pone de manifiesto la importancia de su consideración, especialmente cuando dicha pérdida puede afectar a los rendimientos de algunas producciones animales.

Applying precision feeding techniques in growing-finishing pig operations

The high cost of feed ingredients, the use of non-renewable sources of phosphate and the dramatic increase in the environmental load resulting from the excessive land application of manure are major challenges for the livestock industry. Precision feeding is proposed as an essential approach to improve the utilization of dietary nitrogen, phosphorus and other nutrients and thus reduce feeding costs and nutrient excretion. Precision feeding requires accurate knowledge of the nutritional value of feedstuffs and animal nutrient requirements, the formulation of diets in accordance with environmental constraints, and the gradual adjustment of the dietary nutrient supply to match the requirements of the animals. After the nutritional potential of feed ingredients has been precisely determined and has been improved by the addition of enzymes (e.g. phytases) or feed treatments, the addition of environmental objectives to the traditional feed formulation algorithms can promote the sustainability of the swine industry by reducing nutrient excretion in swine operations with small increases in feeding costs. Increasing the number of feeding phases can also contribute to significant reductions in nutrient excretion and feeding costs. However, the use of precision feeding techniques in which pigs are fed individually with daily tailored diets can further improve the efficiency with which pigs utilize dietary nutrients. Precision feeding involves the use of feeding techniques that allow the provision of the right amount of feed with the right composition at the right time to each pig in the herd. Using this approach, it has been estimated that feeding costs can be reduced by more than 4.6%, and nitrogen and phosphorus excretion can both be reduced by more than 38%. Moreover, the integration of precision feeding techniques into large-group production systems can provide real-time off-farm monitoring of feed and animals for optimal slaughter and production strategies, thus improving the environmental sustainability of pork production, animal well-being and meat-product quality.

Biological impact assessment of nanomaterial used in nanomedicine. Introduction to the NanoTEST project.

Therapeutic nanoparticles (NPs) are used in nanomedicine as drug carriers or imaging agents, providing increased selectivity/specificity for diseased tissues. The first NPs in nanomedicine were developed for increasing the efficacy of known drugs displaying dose-limiting toxicity and poor bioavailability and for enhancing disease detection. Nanotechnologies have gained much interest owing to their huge potential for applications in industry and medicine. It is necessary to ensure and control the biocompatibility of the components of therapeutic NPs to guarantee that intrinsic toxicity does not overtake the benefits. In addition to monitoring their toxicity in vitro, in vivo and in silico, it is also necessary to understand their distribution in the human body, their biodegradation and excretion routes and dispersion in the environment. Therefore, a deep understanding of their interactions with living tissues and of their possible effects in the human (and animal) body is required for the safe use of nanoparticulate formulations. Obtaining this information was the main aim of the NanoTEST project, and the goals of the reports collected together in this special issue are to summarise the observations and results obtained by the participating research teams and to provide methodological tools for evaluating the biological impact of NPs.

Copeptin is associated with kidney length, renal function, and prevalence of simple cysts in a population-based study.

Arginine vasopressin (AVP) has a key role in osmoregulation by facilitating water transport in the collecting duct. Recent evidence suggests that AVP may have additional effects on renal function and favor cyst growth in polycystic kidney disease. Whether AVP also affects kidney structure in the general population is unknown. We analyzed the association of copeptin, an established surrogate for AVP, with parameters of renal function and morphology in a multicentric population-based cohort. Participants from families of European ancestry were randomly selected in three Swiss cities. We used linear multilevel regression analysis to explore the association of copeptin with renal function parameters as well as kidney length and the presence of simple renal cysts assessed by ultrasound examination. Copeptin levels were log-transformed. The 529 women and 481 men had median copeptin levels of 3.0 and 5.2 pmol/L, respectively (P<0.001). In multivariable analyses, the copeptin level was associated inversely with eGFR (β=-2.1; 95% confidence interval [95% CI], -3.3 to -0.8; P=0.002) and kidney length (β=-1.2; 95% CI, -1.9 to -0.4; P=0.003) but positively with 24-hour urinary albumin excretion (β=0.11; 95% CI, 0.01 to 0.20; P=0.03) and urine osmolality (β=0.08; 95% CI, 0.05 to 0.10; P<0.001). A positive association was found between the copeptin level and the presence of renal cysts (odds ratio, 1.6; 95% CI, 1.1 to 2.4; P=0.02). These results suggest that AVP has a pleiotropic role in renal function and may favor the development of simple renal cysts.