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Title: Using single-cell RNA sequencing to investigate mesenchymal cells during renal injury
Author: Smith, James Roderick
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2017
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Renal fibrosis comprises extra cellular matrix (ECM) deposition, distortion of normal tissue architecture, and ultimately progressive renal impairment, and is the shared end-point of many diverse types of renal injury. Myofibroblasts, responsible for ECM deposition, appear in response to injury and are thought to derive from tissue resident perivascular cells, although these mesenchymal cell types are heterogenous and remain poorly understood. I aimed to characterise the mesenchymal perivascular cell contribution to experimental renal fibrosis to better understand this complex process. Renal fibrosis was induced in Pdgfrb-BAC-eGFP mice by Unilateral Ureteric Obstruction (UUO). In this transgenic mouse, cells expressing Platelet-Derived Growth Factor Receptor beta (PDGFR-b), a broad mesenchymal cell marker, also express Green Fluorescent Protein (GFP). Uninjured, day 2 UUO and day 10 UUO kidneys were digested and cells expressing PDGFR-b were sorted individually into wells of 384-well plates before being processed for Single-Cell RNA-Sequencing (scRNA-seq). Bioinformatics analysis was performed to quality control the data and identify clusters of cells expressing similar gene signatures, differentially expressed genes between clusters, marker genes and gene ontology modules unique to clusters and injury timepoints. In total 960 cells were sorted and sequenced, with 817 (85.1%) of these surviving quality control metrics. Using unsupervised clustering, 12 PDGFR-b+ cell clusters were identified, comprising a range of cells with mesenchymal cell type gene signatures, likely subsets of perivascular pericytes, myofibroblasts, mesangial cells and periglomerular vascular smooth muscle cells. Some clusters were unique to injury, including a small subset of Renin 1 expressing cells, while others persisted relatively unchanged throughout injury. In addition subtypes of glomerular epithelial cells, including podocytes and at least two parietal epithelial cell populations expressing varying degrees of podocyte-like and tubular epithelial-like genes, were identified. scRNA-seq applied to mesenchymal cells during the course of experimental renal fibrosis has allowed the identification of a heterogenous range of cell types, cell states and marker genes previously difficult to capture. Further work is required to characterise the functional significance of these findings in vivo.
Supervisor: Henderson, Neil ; Serrels, Alan ; Hughes, Jeremy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: kidney scarring ; myofibroblasts ; pericytes ; extra-cellular matrix ; ECM deposition ; renal fibrosis