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Title: The characterisation of the novel effects of low-iron on renal epithelial cells
Author: Haley, Matthew Alan
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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In mammals, effective acquisition and handling of iron is critical for survival. Consequently, iron handling proteins are exquisitely responsive to changes in iron concentrations. Regulation occurs at both the transcriptional and translational levels. The work described in this thesis focuses on two iron handling proteins that are central to iron regulation and homeostasis, namely divalent metal transporter 1 (DMT1) and transferrin (Tf). Experiments were performed independently to test two aims and accordingly the outcomes of these experiments are described separately. 1. To determine the cellular location of heterologously expressed iron induced (DMT1) splice variants. Two novel DMT1 splice variants, lacking exon 5 (DMT1Δ5) and exons 12(DMT1Δ12) were induced by treating cells with desferrioxamine (DFO), a treatment that lowers the cellular iron concentration. Previously, DMT1Δ5 and DMT1Δ12 have been described in patients with DMT1 mutations, however,these studies did not establish a direct link between mutations, anaemia or splicing. Therefore, as a first step towards understanding their function we aimed to engineer DMT1Δ5 and DMT1Δ12 constructs tagged with mRFP and express them in non-polarised and polarised cell models. Heterologous expression of DMT1 in WKPT cells localised predominantly to structures resembling the late endosomes and lysosomes as previously shown. In contrast, mRFP-DMT1Δ5 localised to the nuclei and led to cell death after 16 hours post transfection in WKPT cells. However, no expression of mRFPDMT1Δ12 was detected in hamster embryonic kidney 293 or WKPT cells. These studies suggest that low-iron induced splicing events may differentially affect subcellular localisation of DMT1 in polarised and non-polarised cells. 2. To determine the mechanism of transferrin uptake into the WKPT renal epithelial cell line. Multi-ligand receptors, megalin/cubilin are suggested to be the mediators of Tf uptake in the proximal tubule. However in mice, the TfR1 has been shown to localise at the apical membrane of the proximal tubule epithelia. Therefore, we aimed to determine the contribution of megalin/cubilin to cellular Tf delivery in the PT. Experiments were performed in vitro, using a cell line (WKPT-0293Cl.2) derived from the proximal tubule of rats. Ligands of megalin/cubilin, receptor associated protein (RAP) and Tf were used for uptake studies. Uptake studies confirmed that when iron is replete, megalin/cubilin are the main mode of Tf (Tfmeg/cub) uptake in PT cells. However, when cellular iron was reduced, a switch to a RAP insensitive receptor (RIR) upregulated Tf (TfRIR)uptake, leading to an increase in intracellular Tf. Further experiments showed that Tf enters a common pathway irrespective of the receptor in which its uptake is mediated. However, TfRIR was retained for longer in comparison to Tfmeg/cub, highlighting a key difference between the two intracellular pathways of Tfmeg/cub and TfRIR. In summary, data suggested that megalin/cubilin are the dominant means by which Tf is endocytosed when iron was replete. However when cellular iron is restricted, TfR1 switches to become the principal receptor for Tf endocytosis.
Supervisor: Ashton, Nick; Smith, Craig Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available