9C.09: TREATMENT WITH CANDESARTAN UNMASKS HIGHER URINARY NOREPINEPHRINE EXCRETION IN WHITE COAT HYPERTENSIVE PATIENTS COMPARED TO HEALTHY NORMOTENSIVE PARTICIPANTS.

OBJECTIVE: White coat hypertensive is a pre-hypertensive state that has been associated with increased sympathetic drive. The objective of the study was to compare the exposure of the kidney to sympathetic nerve activity using urinary normetanephrine (UNMN) as a marker of renal sympathetic exposure in white coat hypertensive (WCH) and healthy normotensive (HN) participants. DESIGN AND METHOD: This was a double-blind randomized placebo-controlled crossover study. WCH were included if office blood pressure was >140/80 mmHg and ambulatory blood pressure <135/85 mmHg and HN if OBP was <140/90 mmHg and ABP <135/85 mmHg Participants were randomized to receive either 16 mg of candesartan or a matched placebo for one week before study day. On the study day systemic and renal hemodynamics as well as plasma norepinephrine and urinary excretion of normetanephrine (measured by LC/MS-MS were measured after one hour of baseline, one hour of lower body negative pressure and one hour of recovery period. Excretion of UNMN was expressed as the total of UNMN excreted during these three hours (cumUNMN). Paired or unpaired t-test were used for comparison. RESULTS: 25 HN and 12 WCH participants were included in the study. Mean age (±standard deviation), BMI were respectively 31.0±10.5 years and 22.0 ± 2.2 Kg/m2 in HN and 40.7±17.8 years and 26.7 ± 6.3 Kg/m2 in WCH.Table 1 Baseline mean blood pressure, plasma noradrenaline and cumulated UNMN during placebo and candesartan(Figure is included in full-text article.)Mean blood pressure was higher during placebo and candesartan in WCH compared to HN. Cumulated UNMN was higher in both groups after candesartan treatment. Cumulated UNMN was higher in WCH than in HN only after candesartan treatment. CONCLUSIONS: Urinary excretion of normetanephrine is increased in WCH compared to HN when treated with candesartan. The increased excretion of uNMN when the renin angiotensin system is blocked might reflect an increased sensitivity of WCH to stress conditions such as orthostatic stress.

Association of urinary calcium excretion with serum calcium and vitamin D levels

Contexte de recherche Mon projet de thèse en médecine concerne l'exploration des facteurs associés à l'excrétion urinaire de calcium durant 24h dans une étude populationnelle suisse. Les données qui ont été utilisées sont issues d'une étude populationnelle connue sous le nom de « Swiss survey on Salt intake », qui a été effectuée entre janvier 2010 et mars 2012 et avait pour but principal d'investiguer la consommation de sel de la population suisse. L'étude a regroupé plusieurs centres d'investigations dans toute la Suisse (11 centres dans 9 cantons), dans trois langues nationales (français, allemand et italien) et concerne des participants âgés de plus de 15 ans. Cette étude avait l'avantage de collecter des données anthropométriques, sanguines, urinaires, de lister les traitements et de poser des questions sur le style de vie (alcool, activité physique, etc.). A notre connaissance, peu d'articles populationnels explorent les facteurs associés à l'excrétion urinaire de calcium durant 24h et notamment l'association de celle-ci avec le calcium corrigé sanguin et la 250H-vitamine D2*3 sanguin chez les hommes et les femmes. Méthodes Après exclusions des données manquantes, 1293 participants sur 1550 ont été retenus pour l'analyse statistique. Celle-ci a été effectuée grâce au logiciel Stata 12. Nous avons utilisé la régression linéaire multiple dont la variable dépendante était l'excrétion urinaire de calcium durant 24h en stratifiant les analyses par sexe (en raison des interactions statistiques significatives entre calcémie et sexe, et entre vitamine D et sexe). Les variables indépendantes comprenaient des variables en lien avec l'excrétion urinaire de calcium (vingt-quatre covariables au total) dont la 250H-vitamine D2+3 (avec la variabilité saisonnière) et la calcémie (calcium sérique corrigé par les protéines). Résultats D'importantes différences entre hommes et femmes ont été observées : une association positive entre l'excrétion urinaire de calcium durant 24h et la calcémie corrigée a été trouvée chez les femmes mais pas chez les hommes. En parallèle, nous relevons une association positive entre l'excrétion urinaire de calcium durant 24h et la 250H-vitamine D2+3 chez les hommes mais pas chez les femmes (liée essentiellement à la présence 250H-vitamine D3 en quantité bien plus importante que la 250H-vitamine D2). Nous soulevons deux hypothèses possibles pour expliquer ces importances différences hommes-femmes. La première hypothèse est l'influence des hormones sexuelles. La seconde hypothèse est la contribution des facteurs diététiques. De nouvelles analyses et de nouvelles études populationnelles sont nécessaires pour confirmer ou infirmer ces hypothèses. Perspectives Une prochaine étape sera d'explorer l'influence de l'excrétion urinaire de sodium, de potassium et d'urée sur les associations observées afin d'étudier une possible influence de l'alimentation sur ces résultats. Une autre étape consistera à doser les hormones sexuelles dans les échantillons de cette étude ou dans ceux d'une autre étude. La prévalence de l'hypercalciurie dans cette étude (9 % chez les hommes et 8.1% chez les femmes) est plus basse que dans d'anciennes études non-populationnelles. Il paraît important de mentionner qu'il existe peu de données sur les normes de calciurie dans la population générale et qu'il n'existe pas de consensus international claire concernant la définition de l'hypercalciurie. Cette prévalence élevée est notamment pertinente dans la cadre des néphrolithiasés, qui sont connues pour être plus fréquentes chez les hommes que chez les femmes. Enjeux Les différences hommes-femmes observées pourraient avoir des implications pour améliorer notre compréhension des mécanismes impliqués dans les lithiases urinaires et dans le risque cardiovasculaire associé ainsi que dans notre compréhension des mécanismes associés à l'ostéoporose.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children : Tracing the complexity of human postnatal steroidogenesis.

CONTEXT: Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by (a) the metabolites of the fetal-placental unit at birth, (b) the fetal adrenal androgens until its involution 3-6 months postnatally, and (c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE: The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS: We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS: 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION: The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.

Isoelectric focusing of urinary metallothionein.

Isoelectric focusing of human urinary metallothionein at a pH range of 4.8 to 7.0 yielded a single protein band with a pI of 5.57 which co-migrated with authentic purified metallothionein I from human liver. Minimum pretreatment of the urine samples (160 ml) was needed. The preparatory steps included sample concentration with the original protein, enriched from 69 +/- 23 micrograms/ml to 2.0 +/- 1.4 mg/ml (+/- SD; n = 9), followed by heat treatment at 80 degrees C for 5 min (2.4 +/- 1.7 mg protein/ml). After focusing, the gels were stained with silver and the lanes were scanned with a laser scanner. Peak areas were used for quantitation with commercial beta 2-microglobulin as a standard. The urinary metallothionein ranged from 1.0 to 2.6 nmol/mmol creatinine, which is comparable with values reached by radio-immunoassay.

Associations of Urinary Uromodulin with Clinical Characteristics and Markers of Tubular Function in the General Population.

BACKGROUND AND OBJECTIVES: Allelic variants in UMOD, the gene coding for uromodulin, are associated with rare tubulointerstitial kidney disorders and risk of CKD and hypertension in the general population. The factors associated with uromodulin excretion in the normal population remain largely unknown, and were therefore explored in this study. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Urinary uromodulin excretion was measured using a validated ELISA in two population-based cohorts that included more than 6500 individuals. The Swiss Kidney Project on Genes in Hypertension study (SKIPOGH) included 817 adults (mean age±SD, 45±17 years) who underwent renal ultrasonography and performed a 24-hour urine collection. The Cohorte Lausannoise study included 5706 adults (mean age, 53±11 years) with fresh spot morning urine samples. We calculated eGFRs using the CKD-Epidemiology Collaboration formula and by 24-hour creatinine clearance. RESULTS: In both studies, positive associations were found between uromodulin and urinary sodium, chloride, and potassium excretion and osmolality. In SKIPOGH, 24-hour uromodulin excretion (median, 41 [interquartile range, 29-57] mg/24 h) was positively associated with kidney length and volume and with creatinine excretion and urine volume. It was negatively associated with age and diabetes. Both spot uromodulin concentration and 24-hour uromodulin excretion were linearly and positively associated (multivariate analyses) with eGFR<90 ml/min per 1.73 m(2). CONCLUSION: Age, creatinine excretion, diabetes, and urinary volume are independent clinical correlates of urinary uromodulin excretion. The associations of uromodulin excretion with markers of tubular functions and kidney dimensions suggest that it may reflect tubule activity in the general population.

Multigenerational effects of the anticancer drug tamoxifen and its metabolite 4-hydroxy-tamoxifen on Daphnia pulex.

Tamoxifen and its metabolite 4-hydroxy-tamoxifen (4OHTam) are two potent molecules that have anticancer properties on breast cancers. Their medical use is expected to increase with the increasing global cancer rate. After consumption, patients excrete tamoxifen and the 4OHTam metabolite into wastewaters, and tamoxifen has been already detected in wastewaters and natural waters. The concentrations of 4OHTam in waters have never been reported. A single study reported 4OHTam effects on the microcrustacean Daphnia pulex. The effects of tamoxifen and 4OHTam over more than two generations are unknown in aquatic invertebrates. The main goal of this study was to assess the long-term sensitivity of the microcrustacean D. pulex over four generations, based on size, reproduction, viability and the intrinsic rate of natural increase (r). Additional experiments were carried out to observe whether the effects of tamoxifen and 4OHTam were reversible in the next generation after descendants were withdrawn from chemical stress (i.e., recovery experiment), and whether the lowest test concentration of each chemical induced toxic effects when both concentrations were combined (i.e., mixture experiments). Our results showed that tamoxifen and 4OHTam induced the adverse effects at environmentally relevant concentrations. Tamoxifen and 4OHTam impaired size, viability, reproduction and the r in four generations of treated D. pulex, but these effects were not clearly magnified over generations. Tamoxifen was more potent than 4OHTam on D. pulex. When used in a mixture, the combination of tamoxifen and 4OHTam induced effects in offspring, whereas no effects were observed when these chemicals were tested individually. In the recovery experiment, the reproduction and size were reduced in offspring withdrawn from chemical exposures. Our results suggested that tamoxifen and its metabolite may be a relevant pharmaceutical to consider in risk assessment.

Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures.

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.

Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection

BACKGROUND: In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model. METHODS: In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed. RESULTS: Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage. CONCLUSIONS: Further development of A1AT as a diagnostic biomarker for BCa is warranted.

A novel metabolite from the Chilean mollusk Siphonaria lessoni

A new and two previously known metabolites possessing a polypropionate carbon skeleton have been isolated from the marine gastropod mollusk Siphonaria lessoni, collected at Chilean coasts. Their structures have been determined by spectroscopical methods.

Reactions of Chlorambucil and its main metabolite, Phenylacetic Acid Mustard, with 2’-deoxyribonucleosides and Calf Thymus DNA

Chlorambucil is an anticancer agent used in the treatment of a variety of cancers, especially in chronic lymphocytic leukemia, and autoimmune diseases. Nevertheless, chlorambucil is potentially mutagenic, teratogenic and carcinogenic. The high antitumor activity and high toxicity of chlorambucil and its main metabolite, phenylacetic acid mustard, to normal tissues have been known for a long time. Despite this, no detailed chemical data on their reactions with biomolecules in aqueous media have been available. The aim of the work described in this thesis was to analyze reactions of chlorambucil with 2’-deoxyribonucleosides and calf thymus DNA in aqueous buffered solution, at physiological pH, and to identify and characterize all adducts by using modern analyzing methods. Our research was also focused on the reactions of phenylacetic acid mustard with 2’-deoxynucleosides under similar conditions. A review of the literature consisting of general background of nucleic acids, alkylating agents and ultraviolet spectroscopy used to identify the purine and pyrimidine nucleosides, as well as the results from experimental work are presented and discussed in this doctoral thesis.

A simple and rapid method for urinary acetone analysis by headspace/gas chromatography

Urinalysis of acetone is important to monitor workers occupationally exposed to acetone and/or isopropanol, as well as in diagnosis of some diseases related to lipid metabolism impairment. This work shows a sensitive, simple and rapid static headspace-gas chromatographic procedure for quantitative determination of acetone in urine. The method was applied to measure acetone in 207 samples from general population volunteers, resulting in a mean level of 1.12 mg/L (± 0.47) and a range of 0.20 - 1.95 mg/L. The method is reproducible and reliable, making it suitable for routine analysis of acetone in urine.