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Title: Investigating the mechanisms of renal fibrosis following ischaemia and reperfusion injury
Authors: Kapoor, Rishab
Issue Date: 2018
Publisher: Newcastle University
Abstract: Ischaemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI) and predisposes to the development of chronic kidney disease (CKD). The role of TGF-β in extracellular matrix deposition and renal fibrosis has been well established. This study was designed to establish an in vitro model of renal tubular IRI, evaluate the role of TGF-β in IRI in human proximal tubular epithelial cells (HKC8 and HK2 cells) and further determine the role of αvβ6 integrin in IRI. Initially an in vitro model of hypoxia and free radical stress by treating HKC8, HK2 and fibroblasts (MRC-5 cells) with CoCl2 and H2O2 respectively was established. These treatments led to pro-fibrotic changes characterised by increased expression of fibrotic marker α-SMA and reduced expression of epithelial cell marker E-Cadherin at mRNA and protein level. Binding of TGF-β to its receptor leads to activation of the kinase ALK5. ALK5 inhibition prevented the changes induced by H2O2 or CoCl2 suggesting the involvement of TGF-β to the cellular response to IRI. To confirm that TGF-β is released after treatment of cells to mimic IRI, media transfer and co-culture studies were performed. These experiments confirmed that bioactive TGF-β was being released. Lastly, the role of αvβ6 integrin was studied post H2O2 or CoCl2 treatment. Expression of αvβ6 integrin was elevated in conditions mimicking IRI in vitro and in biopsy samples acquired from patients with acute tubular necrosis and in mouse kidney following IRI. Knockdown of αvβ6 integrin in HKC8 cells decreased the bioavailability of active TGF-β following CoCl2 or H2O2 treatment and therefore the pro-fibrotic changes that were seen. This study confirms that bioactive TGF-β is produced following IRI and αvβ6 plays an important role in its release.
Description: PhD Thesis
Appears in Collections:Institute of Cellular Medicine

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