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Title: The expression of CX₃CL1 (fractalkine) in renal tubular epithelial cells and the regulation of CX₃CL1 by stimulation of the thromboxane prostanoid receptor
Author: Durkan, Anne Maria
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2012
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Most renal diseases have a common end inflammatory pathway, which is associated with a leukocytic infiltrate. Chemokines are small proteins that are responsible for the chemoattraction of leukocytes into areas of injury or insult. CX3CL1, also known as fractalkine, exists as a transmembrane protein as well as a soluble protein. It acts as a cell adhesion molecule in addition to its chemoattractant properties. This thesis firstly examines the distribution of CX3CL1 in renal tubular epithelial cells (RTEC) and in the second part of the thesis the regulation of CX3CL1 by stimulation of the thromboxane prostanoid (TP) receptor is examined. The localisation of CX3CL1 was initially demonstrated primarily on the apical surface of tubular epithelial cells in human renal biopsy specimens with histological diagnoses of acute tubular necrosis and acute allograft rejection. A cell model was then developed in MDCK cells to examine the distribution more closely. There are a limited number of mechanisms potentially responsible for the trafficking of CX3CL1 to the apical membrane and it was established that N-glycosylation of CX3CL1 is required for its presence on the apical membrane of RTEC. The mobility of CX3CL1 within the cell membrane was next assessed and it was shown to be relatively immobile. We hypothesized that this would promote cell adhesion and indeed further experiments confirmed that CX3CL1 in RTEC does promote adhesion of cells bearing the cognate receptor. Given that CX3CL1 and thromboxane A2 are both found in similar inflammatory conditions, are both present early in the inflammatory process and that stimulation of the TP receptor has been shown to regulate other chemokines, we next evaluated the effect of stimulation of TP on CX3CL1. We found that both total and surface cellular levels of CX3CL1 were reduced following stimulation of TP. A maximal nadir was present after 30-60 minutes and the levels returned to baseline by 4 hours. The mechanism for the loss of CX3CL1 was then assessed. CX3CL1 is known to recycle between the cell surface and an internal compartment. No effect of TP stimulation was seen on the endocytosis or exocytosis of CX3CL1. Stimulation of TP was however, shown to stimulate tumour necrosis factor-a converting enzyme (TACE) via ERK phosphorylation. TACE inducibly cleaves CX3CLI, releasing the soluble chemokine. TACE siRNA was used to knock down TACE gene expression and this prevented the loss of cellular CX3CL1, confirming that TP stimulation induces TACE cleavage of CX3CL1. T he results of further experiments are discussed in the discussion chapter.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available