Wigmore, Stephen

Ross, James

O'Neill, Stephen

Medical Research Council (MRC)

2017-02-22T16:06:16Z

2017-02-22T16:06:16Z

2015-11-27

Kidney transplantation is the gold standard treatment for end-stage renal disease. Renal ischemia-reperfusion injury is an unavoidable consequence of the transplantation procedure and is responsible for delayed graft function and poorer long-term outcomes. Pharmacological inhibition of heat shock protein 90 is a preconditioning strategy that has previously been shown to reduce renal ischemia-reperfusion injury. However, the clinical application of heat shock protein 90 inhibitors is limited by their toxicity profile and the exact mechanisms of protection conferred are unknown. The aims of this thesis were to establish mechanisms of protection offered by these drugs and investigate a less toxic analogue that has the potential to be safely translated into human studies. AT13387 is a novel small molecule heat shock protein 90 inhibitor with a low toxicity profile, which is being evaluated in phase II studies in oncology and therefore has excellent translational potential in the context of transplantation. Heat shock protein 90 inhibition up-regulates protective heat shock proteins (especially heat shock protein 70) and potentially down-regulates NF-ҡB activity by disruption of the IҡB kinase complex. Toll-like receptor 4 is a further regulator of NF-ҡB activity and studies have suggested that Toll-like receptor 4 plays a dominant role in mediating kidney damage following ischemia-reperfusion injury. To explore potential molecular mechanisms of protection, human embryonic kidney cells were pre-treated with AT13387 and exposed to endotoxin-free hyaluronan to stimulate sterile Toll-like receptor 4-specific NF-ҡB activation. AT13387-treatment resulted in breakdown of IҡB kinase, which abolished Toll-like receptor 4-mediated NF-ҡB activation by hyaluronan. Inhibition of autophagy prevented IҡB kinase-α degradation by heat shock protein 90 inhibition and resulted in regain of NF-ҡB activity by hyaluronan. In subsequent investigations, AT13387 decreased pro-inflammatory cytokine release following hyaluronan stimulation and increased cell viability in an in vitro model of oxidative stress. In mice, AT13387 induced heat shock protein 70 expression in the kidney. AT13387 pre-treatment then significantly reduced kidney injury following renal ischemia-reperfusion injury. In contrast, in severe combined immunodeficient mice, AT13387 no longer reduced kidney injury from renal ischemia-reperfusion injury. This emphasises the potential importance of the adaptive immune system in the protective effect of this agent. This resonates with reports of heat shock protein 70 up-regulation in the context of heat preconditioning, which leads to renal protection from renal ischemia-reperfusion injury that is lymphocyte-dependent. Secondary lung injury is an additional consequence of renal ischemia-reperfusion injury. In further experiments, pre-treatment with AT13387 again did not reduce kidney injury following renal ischemia-reperfusion injury in severe combined immunodeficient mice. However, AT13387 did reduce secondary lung injury. This lung protective effect may have been related to heat shock protein 70 up-regulation in the lungs by AT13387. A rationale for enhancing recovery, following renal ischemia-reperfusion injury, by inhibiting heat shock protein 90 was then sought. This investigation was undertaken in order to broaden the range of the available therapies to a wider group of patients including renal transplant recipients. AT13387 pre-treatment of the recipient mice preceded an isograft renal transplantation with a kidney harvested from a treatment naive mouse and cold stored for 4 hours. Although a significant reduction in tubular necrosis was not demonstrated following AT13387 treatment, the feasibility of the treatment strategy was demonstrated and interestingly lung injury secondary to transplantation was reduced. This thesis therefore highlights AT13387 as a new agent with the potential of reducing kidney injury and secondary lung injury following renal ischemia-reperfusion injury. The findings also demonstrate that the mechanisms of protection offered by this drug may involve the adaptive immune system. In addition to the induction of heat shock protein 70 expression in the kidney and repression of Toll-like receptor 4-mediated NF-ҡB signalling through breakdown of IҡB kinase.

http://hdl.handle.net/1842/20410

en

The University of Edinburgh

O'Neill S, Ross JA, Wigmore SJ, Harrison EM. The role of heat shock protein 90 in modulating ischemia-reperfusion injury in the kidney. Expert Opinion on Investigational Drugs 2012; 21 (10): 1535-1548.

O'Neill S, Hughes J. Heat-shock protein-70 and regulatory T cell-mediated protection from ischemic injury. Kidney International 2014; 85 (1): 5-7.

O'Neill S, Harrison EM, Ross JA, Wigmore SJ, Hughes J. Heat-Shock Proteins and Acute Ischaemic Kidney Injury. Nephron Experimental Nephrology 2014; 126 (4): 167-174.

O'Neill S, Gallagher K, Hughes J, Wigmore SJ, Ross JA, Harrison EM. Challenges in early clinical drug development for ischemia-reperfusion injury in kidney transplantation. Expert Opinion on Drug Discovery 2015; 10 (7): 753-762.

O'Neill S, Humphries D, Tse GH, Marson LP, Dhaliwal K, Hughes J, Ross JA, Wigmore SJ, Harrison EM. Heat shock protein 90 inhibition abrogates TLR4-mediated NF-κB activity and reduces renal ischemia-reperfusion injury. Scientific Reports 2015; 7 (5): 12958.

O’Neill S, Hughes J, Ross JA, Wigmore SJ, Harrison EM. Heat shock protein 90 inhibition abrogates TLR4-mediated NF-κB activity and reduces renal ischemia-reperfusion injury. British Journal of Surgery 2013; 100 (S7): 2.

O’Neill S, Hughes J, Ross JA, Wigmore SJ, Harrison EM. Heat shock protein 90 inhibition with AT13387 abrogates TLR4-mediated NF-κB activity and protects from oxidative stress. Transplant International 2014; 27 (S1): 4.

Attribution-NonCommercial-ShareAlike 4.0 International

http://creativecommons.org/licenses/by-nc-sa/4.0/

kidney

ischemia

transplant

Role of heat shock protein 90 in modulating ischemia-reperfusion injury in the kidney

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy