Abstracts oral sessions
Background: Maternal stress affects fetal development. Fetal growth retardation and pre-eclampsia share a common pathogenesis and copeptin, a novel stress marker, is found elevated in pre-eclampsia. The aim of our study was to establish reference intervals for serum-copeptin during pregnancy and to test our hypothesis that maternal serum-copeptin is elevated in women with fetal growth retardation.
Methods: A nested case-control study. Copeptin levels were determined in maternal serum at gestational weeks 12 and 19 by using a Copeptin ultra-sensitive Kryptor kit (BRAHMS). Cases were pregnancies later developing fetal growth retardation (N=39). Controls were normal pregnancies (N=119). Reference ranges were calculated as 95% prediction intervals and presented as anti-logarithm with 90% confidence intervals. Paired and unpaired t-test was performed to test the null-hypothesis of no difference in serum-copeptin levels within and between groups. Odds ratios were calculated for maternal characteristics and risk of fetal growth retardation.
Results: Reference intervals for copeptin in normal pregnancies were 1.24-5.51 pmol/L (90% confidence intervals on upper and lower limit 1.13-1.37 and 5.00-6.08 pmol/L) at gestational week 12, and 1.30-5.09 pmol/L (90%
confidence intervals 1.19-1.42 and 4.65-5.57 pmol/L) at gestational week 19. Copeptin levels decreased from week 12 to 19 in cases (p=0.02), whereas no significant decrease was observed in controls (p=0.61). No difference was found in copeptin levels comparing cases with controls in gestational week 12 (p=0.10) and week 19 (p=0.81).
Conclusions: Copeptin did not add predictive information on fetal growth retardation.
O01.03 Anders Jorsal CARDIAC EFFECTS OF GLP-1 TREATMENT IN PATIENTS WITH CHRONIC HEART FAILURE
A. Jorsal1,2, H. Wiggers1,2, P. Holmager3, R. Nielsen1,2, T. Welløv Boesgaard4, A. Kumme5, J. Møller5, L. Videbæk5, C. Kistorp3,
I. Gustafsson6, L. Tarnow4,7,8, A. Flyvbjerg7,9
1Department of Cardiology, Aarhus University Hospital, 2Department of Clinical Medicine, Faculty of Health, Aarhus University, 3Department of Endocrinology and Internal Medicine, Herlev University Hospital, 4Steno Diabetes Center, 5Department of Cardiology, Odense University Hospital,
6Department of Cardiology, Hvidovre University Hospital, 7Faculty of Health, Aarhus University, 8Hilleroed University Hospital, 9Department of
Endocrinology and Internal Medicine, Aarhus University Hospital Background: Glucagon-like peptide 1 (GLP-1) is a naturally existing
hormone, which is a part of the incretine system. GLP-1 stimulates insulin production and inhibits glucagon-excretion from the pancreas, thereby reducing blood sugar levels. Moreover, a beneficial effect of GLP-1 on cardiac function has been suggested in both diabetic and non-diabetic patients. Liraglutide (Victoza®) is a GLP-1-analogue developed for treatment of type 2 diabetes (T2D). The impact of liraglutide on cardiac function has not previously been investigated in patients with chronic heart failure (CHF).
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Hypothesis: Liraglutide treatment for 24 weeks improves LVEF in CHF patients with and without T2D compared to placebo treatment.
Design and methods: An investigator initiated, multi-centre, randomised, double blind, parallel, placebo controlled intervention trial. In total, 240 CHF patients (patients with T2D and patients without diabetes 1:1) with left ventricular ejection fraction < 45% will be randomised to either
subcutaneous injection of liraglutide 1.8 mg or matching placebo once daily for 24 weeks. The effect of liraglutide on left ventricular systolic function will be evaluated by advanced echocardiography, including 3-dimensional contrast echocardiography.
Status (1 September 2013):
Randomised: 134 (screened:255) Ischemic heart disease: 68%
Diabetes: 33%
Adverse events: 168 Serious adverse events: 18
Perspectives: Potentially liraglutide can improve heart function sub-stantially, thus changing the prognosis of CHF patients worldwide.
O01.04 Vibeke Secher Nielsen
FUNCTIONAL CHARACTERISTICS OF PUTATIVE CA2+-ACTIVATED CL- CHANNELS - BESTROPHINS AND TMEM16A - IN RAT MESENTERIC SMALL ARTERIES
V.S. Dam1, T. Brøgger2, D.B. Bødtkjer1, C. Aalkjær1, V. Matchkov1
1Department of Biomedicine, Faculty of Health, Aarhus University,
2Department of Clinical Medicine, Faculty of Health, Aarhus University The presence of Ca2+-activated Cl- current (ICl(Ca)) in vascular smooth
muscle cells (VSMCs) is well established and has been suggested to be important for synchronized rhythmic contraction, i.e. vasomotion. The molecular background for this membrane conductance is, however, elusive. Bestrophins (Best) and TMEM16 proteins are the most prominent candidates for this role. We have previously characterized two distinct ICl(Ca)
in VSMCs: the cGMP-dependent ICl(Ca) current and the "classical" ICl(Ca). Best-3 or TMEM16A were knocked down in vivo in rat mesenteric small arteries using siRNA. Knockdown (KD) was confirmed at mRNA and protein levels. The ICl(Ca) were measured by patch clamp. Arterial contraction in vitro was tested using isometric myography.
Best-3 KD induced secondary reduction of Best-1 and -2 expression, while TMEM16A expression was not affected. TMEM16A KD reduced the
expression of Best. In contrast to Best-3 KD, which only suppressed the cGMP-dependent ICl(Ca), TMEM16A KD suppressed both ICl(Ca) currents. Co-immunoprecipitation revealed a physical interaction between TMEM16A and Best-3. Best-3 KD was without an effect on arterial contraction.
TMEM16A KD reduced membrane depolarization, [Ca2+]i and tension increase in response to agonist stimulation. This can be partially explained by secondary reduction in the expression of L-type Ca2+ channels. Vaso-motion was significantly suppressed after both TMEM16A and Best KDs.
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Bests and TMEM16A are both important for vasomotion in mesenteric small arteries, but only TMEM16A is involved in agonist-induced contraction.
Regulatory interactions between these proteins occur at the expressional and functional levels.
O01.05 Mie Rostved Rasmussen
IDENTIFICATION OF SUBSTRATE SEQUENCE MOTIFS IMPORTANT FOR ADAM17-MEDIATED SHEDDING OF CD163 AND TNF-ALPHA
M.R. Rasmussen1, A. Etzerodt1, P. Svendsen1, A. Chalaris2, J. Schwarz2, I.
Galea3, H.J. Møller4, S.K. Moestrup1
1Department of Biomedicine, Aarhus University, Denmark, 2Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany,
3Clinical & Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK, 4Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark
Upon inflammatory stimulation, the human macrophage membrane proteins CD163 and pro-tumor necrosis factor-alpha (proTNF-α) are subjected to ectodomain cleavage by the metalloprotease ADAM17. The resulting soluble fragment of proTNF-α is termed TNF-α, and it is a potent pro-inflammatory cytokine. The cleaved ectodomain of the haptoglobin-hemoglobin receptor CD163, termed sCD163, has an unknown function.
Inspection of the amino acid sequence of the juxtamembrane domain of human proTNF-α and CD163 revealed a similar palindromic motif (Arg-Ser-(Ser)-Ser-Arg). In the present study, site-directed mutagenesis
experiments demonstrated that these sequences are essential for shedding of proTNF-α and CD163. Mouse CD163 lacks this palindromic sequence, and this protein was shown to be resistant to ADAM17-dependent ectodomain cleavage. Upon ADAM17-dependent stimulation, a soluble fragment resulting from mouse CD163 was, however, detected when the palindromic sequence was knocked in in the juxtamembrane domain of mouse CD163, thus further underscoring the importance of this sequence.
In conclusion, the present study identified a sequence essential for shedding of proTNF-α and CD163 by ADAM17. The study also
demonstrated distinct differences in terms of regulation of CD163 in human and mouse. In humans, proinflammatory-induced shedding of the CD163 ectodomain leads to a down-regulation in surface-bound CD163 and, hence, the capacity for haptoglobin-hemoglobin complex internalization during inflammatory conditions is reduced.
O01.06 Pauline de Bruijn
THE THICK ASCENDING LIMB AS A MAJOR SITE FOR FUROSEMIDE-INDUCED URINARY ACIDIFICATION
P.I.A. de Bruijn
Department of Biomedicine, Aarhus University
Furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide causes urinary acidification and metabolic alkalosis.
This is explained by an increased Na+ load to the distal tubule, which facilitates H+ secretion via the apical H+-ATPase in α-intercalated cells. The direct role of the TAL on the urinary acidification, however, has never been
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investigated. Here we measured pHi in single perfused mouse mTALs with BCECF-AM. Interestingly, luminal furosemide (100 µM) caused a major, stable and reversible intracellular alkalization both in HEPES (0.33 ± 0.04, n=7) and CO2/HCO3--buffered conditions (0.14 ± 0.03, n=5). This
alkalization likely indicates increased H+ excretion from the mTAL cytosol.
Intriguingly, the furosemide-induced alkalization was completely blocked by 1 mM luminal amiloride that fully inhibits the apical NHE3 antiporter. This was confirmed with the NHE3 specific inhibitor NHE#4167. Basolateral amiloride did not affect the alkalization. Thus, furosemide likely causes a NHE3-dependent secretion of H+ to the lumen. To investigate this, we measured the pHo of the tubular lumen with BCECF acid. Furosemide indeed caused a reversible luminal acidification from pH 6.92 ± 0.04 to 6.46 ± 0.03 (n=5) providing direct evidence for this suggested mechanism.
In contrast, luminal amiloride alkalinized the lumen. The increased NHE3 activity induced by furosemide is likely caused by a decrease of [Na+]i. Indeed, mTALs loaded with CoroNa Green showed furosemide-induced drop in [Na+]i. These results revise the mechanistic understanding of furosemide-induced urinary acidification and prove that furosemide stimulates H+ secretion in the TAL.
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