Dietetics, Nutrition and Biological Sciences
Permanent URI for this collectionhttps://eresearch.qmu.ac.uk/handle/20.500.12289/23
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Item Conditional deletion of Hsd11b2 in the brain causes salt appetite and hypertension(American Heart Association, 2016-03-07) Evans, Louise C.; Ivy, Jessica R.; Wyrwoll, Caitlin; McNairn, Julie A.; Menzies, Robert I.; Christensen, Thorbjørn H.; Al-Dujaili, Emad A. S.; Kenyon, Christopher J.; Mullins, John J.; Seckl, Jonathan R.; Holmes, Megan C.; Bailey, Matthew A.Background—The hypertensive syndrome of Apparent Mineralocorticoid Excess is caused by loss-of-function mutations in the gene encoding 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), allowing inappropriate activation of the mineralocorticoid receptor by endogenous glucocorticoid. Hypertension is attributed to sodium retention in the distal nephron, but 11βHSD2 is also expressed in the brain. However, the central contribution to Apparent Mineralocorticoid Excess and other hypertensive states is often overlooked and is unresolved. We therefore used a Cre-Lox strategy to generate 11βHSD2 brain-specific knockout (Hsd11b2.BKO) mice, measuring blood pressure and salt appetite in adults.Item Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion(2009-04) Al-Dujaili, Emad A. S.; Mullins, L. J.; Bailey, M. A.; Kenyon, C. J.Background: Clinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases. Methods: Antibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones. Results: Cross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R2 = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice. Conclusion: We describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia. 2009 Elsevier Inc. All rights reserved.