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Title: Investigating miR-214 as a pro-fibrotic mediator in renal fibrosis and its potential as a therapeutic target via effective in vivo modelling of chronic kidney disease
Author: O'Sullivan, James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Chronic kidney disease (CKD) is a prevalent health condition in the UK and worldwide. CKD is defined as the progressive decline of renal filtrative capacity over time. High rates of cardiovascular dysfunction and related mortality are observed in patients with CKD. Renal fibrosis is the final common pathway though which all CKD, regardless of the initiating insult. Renal fibrosis, defined as excess collagenous matrix deposition that restricts renal filtrative capacity, is a complex process involving multiple cell types, biological pathways, cytokines and other pro-fibrotic factors such as microRNAs (miRNAs). miRNAs are small non-coding RNA which post-transcriptionally regulate gene expression via inhibition of target mRNA translation. MicroRNA-214-3p (miR-214) has been proposed to be a key pro-fibrotic miRNA in renal fibrosis. Increased miR-214 expression is found in CKD patient urine and kidney biopsies, correlating with degree of fibrosis, mirroring the increase observed in several in vitro and in vivo models. Knockdown of miR-214 expression in the unilateral ureteral obstruction (UUO) model of renal fibrosis resulted in an 86% reduction in fibrosis, although the mechanism behind this remained largely unknown. Therefore, the hypothesis presented in this thesis is that miR-214 functions as a pro-fibrotic miRNA in renal fibrosis and effective in vivo modelling will allow for the investigation of its mechanism and assessment of its potential as a therapeutic target in progressive chronic kidney disease. The aims of this thesis are as follows. Firstly, to profile the refined mouse subtotal nephrectomy (STNx) model as a progressive model of CKD. Secondly, to evaluate an anti-miR-214 compound in vitro. Thirdly, to assess the therapeutic potential of antimiR- 214 as an intervention in STNx. Fourthly, to profile the miRNA and gene expression changes observed in individual renal cell populations in UUO and reversible UUO (rUUO). Lastly, to use the generated data to explore potential profibrotic mechanisms of miR-214 in renal fibrosis on a cell-population specific basis. Subtotal nephrectomy is an in vivo model of CKD, which, unlike UUO, allows for the measurement of clinically-relevant renal functional parameters, produces glomerulosclerosis and cardiovascular dysfunction, and has a history of successful translation of findings to clinical practice. The refined subtotal nephrectomy (STNx) 10-week model presented here attempts to overcome downsides in the traditional model; performed as a single-surgery with improved survival, animal welfare and consistent outcomes. The STNx model was fully characterised and then used to assess the effects of miR-214 inhibition (via anti-miR) in this more translatable model of CKD. The anti-miR-214 compound, provided by Regulus Therapeutics, was first assessed in vitro, where its affinity for miR-214 was found to be high and a reduction in profibrotic gene expression was observed in TGFβ-stimulated renal fibroblasts. Intervention with anti-miR-214 in STNx was initiated at 6-weeks post-STNx to assess anti-miR-214 efficacy in a situation where renal fibrosis and dysfunction were already present, as diagnosis and therapeutic intervention in CKD usually do not occur until renal fibrosis and dysfunction are already present. Here, anti-miR-214 intervention did not produce any significant changes across a number of renal and cardiovascular parameters measured compared to control anti-miR. Lack of efficacy may have been due to the timing of the intervention, lack of anti-miR penetration to the appropriate cell types, or lack of efficacy of anti-miR-214 in STNx in general. As miR-214 is known to have diverse and even opposing roles in different cell types and pathologies, elucidating the expression of miR-214 in renal cell types may be critical to understanding its mechanisms in renal fibrosis. In order to address this, the expression of miR-214 was examined in a cell specific and temporal manner using the reversible UUO (rUUO) experimental model. This allowed for analysis of renal injury and resolution of renal injury. From the rUUO model, 4 cell populations known to play a role in renal fibrosis and injury resolution were FACS sorted and underwent RNA sequencing for miRNA and gene expression. miR-214 was found to be significantly enriched in Pdgfrβ+ (myofibroblast-like) cells in the kidney in comparison to proximal tubular epithelial cells, endothelial cells and tissue-resident macrophages, indicating myofibroblasts are the primary site of mechanistic interest. Bioinformatic analysis revealed several predicted miR-214 targets in Pdgfrβ+ cells, indicating miR- 214 may act as an anti-apoptotic and/or pro-proliferative factor in these cells in the context of renal fibrosis. In conclusion, the development and profiling of STNx provided a valuable platform for the assessment of anti-miR-214 as a therapeutic intervention in CKD, allowing the measurement of a variety of clinically-relevant parameters including renal fibrosis. Anti-miR-214, despite displaying efficacy in vitro, did not ameliorate any renal or cardiovascular phenotypes induced by STNx. Four cell populations relevant to renal fibrosis were sorted from rUUO kidneys and RNA-sequencing carried out to discover the gene and miRNA expression profiles in these cell types in renal injury and injury resolution. miR-214 was found to be significantly enriched in Pdgfrβ+ (myofibroblastlike) cells. Bioinformatic analysis of miR-214 targets in this cell population suggests miR-214 may have an anti-apoptotic and/or pro-proliferative role.
Supervisor: Denby, Laura ; Baker, Andrew Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: chronic kidney disease ; miRNAs ; fibrosis ; miR-214 ; unilateral ureteral obstruction ; UUO model ; STNx