PhD ThesisPeople 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
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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