Background: Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes abnormally permeable, leading to the clinical triad of proteinuria, massive oedema, and hypoalbuminaemia. Historically, NS has been thought to result from dysregulation of the immune system, although recent evidence suggests the glomerular podocyte plays a central role in disease pathogenesis. Children with NS are generally treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids which are activating ligands for the glucocorticoid receptor (GR) transcription factor. A major factor limiting the clinical utility of these agents is the marked variation observed in response to treatment. Although Gc-therapy has been the cornerstone of NS management for several decades, the mechanism of action, and target cell, remain poorly understood. Hypothesis and aims: The central hypothesis for this thesis states that glucocorticoids act directly on the podocyte to produce clinically useful effects without involvement of the immune system. Findings: Using a wild-type human podocyte cell line, I demonstrated that the basic GR-signalling mechanism is intact in the podocyte, and that glucocorticoids produce a direct, protective effect on the podocyte without immune cell involvement, by using electrical resistance across a podocyte monolayer as a surrogate marker for barrier integrity. To understand potential mechanisms underpinning this direct effect I defined the podocyte GR cistrome (using a combination of chromatin immunoprecipitation followed by massively parallel DNA sequencing and transcriptomic analysis) as well as total cell proteomics. Subsequent gene ontology analysis revealed that Gc treatment had prominent effects on podocyte motility, and these findings were validated with live-cell imaging. To gain mechanistic insight, I investigated the role of the pro-migratory small GTPase regulator Rac1, and demonstrated that treatment with Gc reduced Rac1 activity. Furthermore, the Rac1 inhibitor EHT 1864 had a direct, protective effect on the podocyte. To create a model to study the role of podocyte GR in vivo I generated a mouse line with a podocyte-specific GR deletion. Impact: Gc exposure produces potentially clinically-relevant effects directly on the podocyte, and Gc-induced podocyte hypomobility may underlie the clinical efficacy of these agents. Future animal studies investigating the consequences of GR deletion in the podocyte and the anti-proteinuric effects of Rac1 inhibition are warranted.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:680009 |
| Date | January 2016 |
| Creators | Mccaffrey, James |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/podocytespecific-glucocorticoid-effects-in-childhood-nephrotic-syndrome(559e1d71-6e81-46e5-84ae-7c836e4de042).html |
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