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Title: Pharmacological strategies to reduce ischemia/reperfusion injury in kidney transplantation
Author: Harrison, Ewen M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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I investigated the influence of the phosphatidylinositol 3-kinase (PI3K)Akt pathway on HSF1 activation status. Despite effecting significant up-regulation of the PI3k/Akt pathway with insulin and insulin-like growth factor-1 (IGF-1), I did not demonstrate any change in the HSF1 trimerisation state, DNA-binding ability or nuclear localisation in renal adenocarcinoma cells (ACHN). Following treatment with insulin, a 5-fold increase in heme oxygenase-1 (HO-1) mRNA and a 4-fold increase in protein expression were observed in ACHN cells; insulin-induced HO-1 expression was also demonstrated in mouse primary tubular epithelial cells. The induction of HO-1 in ACHNs was blocked by actinomycin D and cycloheximide and was abolished by the PI3K inhibitor, LY294002, but not by the inactive analogue, LY303511. Over-expressing a dominant-negative form of Akt abrogated the HO-1 inducing effects of insulin, whereas cells transfected with a constitutively active Akt construct demonstrated an increase in HO-1 promoter activity and protein expression. The transcription factor NF-E2-related factor-2 (Nrf2) was found to translocate to the nucleus following insulin treatment in a PI3K-dependent manner. Pre-treatment with Nrf2 small-interfering RNA (siRNA) abolished insulin-induced HO-1 induction. Thus, insulin induces HO-1 mRNA and protein expression in renal cells in a PI3K/Akt and Nrf2 dependent manner. Heat shock protein 90-binding agents (HBAs) are known to up-regulate Hsps and confer cellular protection. I examined the ability of HBAs to protect the kidney in a model relevant to transplantation. Hsp70 gene expression was increased 30-40 times in ACHN cells treated with HBAs and trimerisation and DNA-binding of HSF1 was demonstrated. A 3 fold and 2 fold increase in Hsp70 and Hsp27 protein expression, respectively, was found in ACHNs treated with HBAs. HBAs protected ACHN cells from an H2O2-mediated oxidative stress and HSF1 siRNA abrogated HBA-mediated Hsp induction and protection. In vivo, Hsp70 was up-regulated in the kidney, liver, lungs and heart of HBA-treated mice. This was associated with a functional and morphological renal protection from IRI. Therefore, HBAs may be useful in reducing transplant-associated kidney injury.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available