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Title: The role of 11β-hydroxysteroid dehydrogenase type 1 in wound healing in uraemia
Author: Duraisingham, Sai Krishna
ISNI:       0000 0004 9355 6103
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2020
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People with Chronic Kidney Disease (CKD), advanced age and systemic cortisol excess demonstrate skin specific similarities including poor wound healing. There is accumulating evidence that patients with advanced kidney disease age prematurely. Studies have demonstrated the cortisol producing 11b-Hydroxysteroid Dehydrogenase Type 1 (11b-HSD-1) enzyme activity is increased in aged skin. We hypothesized the same should be true in the uraemic state. Skin specific 11b-HSD-1 and therefore cortisol excess may be the unifying cause for the skin phenotype and poor healing seen in CKD, advanced age and systemic cortisol excess. The aims of this thesis were to investigate the presence of 11b-HSD-1 and 11b-HSD- 2 within rodent skin with and without uraemia as well as determining the activity of 11b-HSD-1 and to elucidate the role it may play in wound healing. Finally, the efficacy of an 11b-HSD-1 inhibitor in healing was assessed as a potential future therapeutic target. In vitro studies which included Lactate Dehydrogenase (LDH), cell viability, scratch assays and cortisol Enzyme Linked Immunosorbent Assay (ELISA) were conducted using 2 different types of primary skin cultures with the uraemic toxins Indoxyl Sulphate (IS) or p-Cresol (PC) or the specific 11b-HSD-1 inhibitor emodin. An in vivo model of skin wounding was developed in male Wistar rats rendered uraemic by an adenine supplemented diet. Healing was measured in uraemic and non-uraemic control rats at baseline, with the application of topical emodin or with systemic emodin administration. Skin samples were processed ex vivo to extract protein for use in immunoblotting or Ribonucleic Acid (RNA) for Polymerase Chain Reaction (PCR) and a wound healing PCR array. Immunohistochemistry was completed for selected skin samples as were corticosterone (cortisol equivalent in rats) ELISA. The presence of 11b-HSD-1 and 11b-HSD-2 in Human Dermal Fibroblasts (HDF) and Human Epidermal Keratinocytes (HEK) was confirmed by western blotting. No differences in LDH activity, cell viability or cortisol production with the administration of IS, PC or emodin could be detected. Scratch assays utilising uraemic 6 toxins in isolation did not demonstrate a significant delay in wound healing. An improvement in healing using emodin in isolation could also not be demonstrated. Contrary to the in vitro exposure to solitary uraemic toxins, the in vivo findings consistently demonstrated a delay in wound healing in uraemia and the beneficial effect of emodin on healing when administered systemically. This could not be attributed to changes in 11b-HSD-1 or 11b-HSD-2 Messenger RNA (mRNA), protein expression or enzyme activity with the techniques employed. To elucidate a definitive role of 11b-HSD-1 would require the use of radioactive isotopic activity assay and the use of a skin specific 11b-HSD-1 Knock-Out (KO) mouse model. It would be most useful to reproduce this work in human skin, comparing uraemic patients to healthy live kidney donors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available