A single-cell transcriptome atlas of mouse glomerulus
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ABSTRACT: Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. As yet, it remains unknown to what extent cellular heterogeneity exists within healthy glomerular cell populations. Here, we used nanodroplet-based, highly parallel transcriptional profiling to characterize the cellular content of purified wildtype mouse glomeruli. Unsupervised clustering of 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells, which can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry stainings obtained from the Human Protein Atlas. Subclustering of glomerular endothelial cells revealed a subset of activated endothelium, expressing marker genes related to endothelial proliferation. Additionally, the podocyte population could be divided in three different subclusters. In conclusion, our study comprehensively characterizes gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.
Project description:Background: The kidney glomerulus is a specialized capillary bed that is involved in urine production and blood pressure control. Glomerular injury is a major cause of chronic kidney disease, a widespread epidemic without therapeutic options. Single-cell transcriptomics have radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single cell kidney studies due to their paucity and intractability. Methods: We used single-cell RNA sequencing to comprehensively characterize the different types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, CD2AP deficiency). Results: All cell types in the glomerulus were identified using unsupervised clustering analysis. We identified novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arteriole, parietal epithelial cells, and three different types of endothelial cells. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conclusions: We have generated a comprehensive high-resolution single-cell transcriptomic profile of the kidney glomerulus from healthy and injured mice. The dataset provides a useful resource for identifying novel disease-related genes and pathways.
Project description:Renal blood filtration occurs in a functional unit called the glomerulus. The resident cell types comprise the filtration barrier, namely podocytes, mesangial cells and glomerular endothelial cells. Here we introduce a glomerular cell isolation protocol, which enables the separation of these three cell types in sufficient amounts and purity to allow detailed protein-biochemical investigations. We demonstrate that the expression of fluorescent transgenes in glomerular cells can result in proteome artifacts. We show that different mouse strains have different glomerular cell type proteomes. Further, we demonstrate the power of the technique to identify new glomerular cell type-enriched proteins and demonstrate the potential of this globally applicable technique in the dissection of cell-specific disease responses and intra-glomerular cell-type crosstalk.
Project description:to compare the incremental advances that each cell type brings to the phenotype of the others in the glomerulus we compared TGCs (triculture), BGCs (co-culture), and plate cultures (monocultures) The function of the glomerulus depends on complex cell-cell/matrix interactions and replication of this in vitro would aid biological understanding in both health and disease. Previous models have not fully reflected all cell types and interactions present. We report here the development of a microphysiological system which contains all resident renal cell types in an anatomically relevant manner. We demonstrate functionally appropriate retention of albumin in the system along with providing a detailed transcriptomic analysis of the contributing biology of each cell type. The important role of mesangial cells is shown in promoting the health and maturity of the other cell types. AdditionallyAdditionally, we demonstrate that glomerular cells in simple 2D culture exhibit a state more reflective of human disease than previously recognised. This in vitro model will expand our capability to investigate glomerular biology in a more translatable manner by the inclusion of the important mesangial cell compartment.
Project description:Glomerular diseases are most common cause of end stage kidney disease (ESKD). Although renin angiotensin system inhibitors are effective for progression of chronic kidney disease, The number of ESKD increases in the world. To identify a new candidate drug for glomerular disease, differentially expressed genes are searched by glomerular RNA microarray.
Project description:The objective of this study is to create an encyclopedia of all genes expressed in the glomerular endothelial cell under normal and diabetic conditions. We utilized Tie2-GFP transgenic mice to mark cells of the glomerular endothelium. To induce diabetic nephropathy (DB), a genetic model of DB, BKS.Cg-m +/+ Leprdb/J from Jax laboratories was used. We utilized fluorescent activated cell sorting (FACS) to isolate glomerular endothelial cells from normal and diabetic mice. The RNAs from these samples were isolated and utilized to hybridize to microarrays, which offers a powerful, efficient and effective method for the creation of a gene expression atlas. Microarrays were used to identify the transciptional differences that occur in the glomular endothelium of a diabetic mouse. Diabetic and control mice carrying the Tie2-GFP transgenic were utilized to isolate the endothelial cells from the adult glomerulus. The endothelial cells were isolated from the glomerulus using FACS. RNA was isolated and the gene expression profiles were determined by microarrays.
Project description:Here, we applied single-cell RNA seq to uncover the heterogeneity of endothelial cells (ECs) under different stiffness and TGF-β induction. We identified three distinct EC clusters that was consistenyl showed in all different conditions . Further subclustering analysis revealed finer heterogeneity which were differentially impacted by stiffness and TGF-β.
Project description:We characterize the gene expression changes which occur in the mouse glomerular podocyte, mesangial, and endothelial cells between control mice and mutant mice which are missing two copies of Fyn-proto oncogene (Fyn) and one copy of CD2-associated protein (CD2AP) in a mouse model of FSGS.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. Tie2-GFP transgenic mice were utilized to isolate the endothelial cell population from the glomerulus of adult kidneys. The glomerular endothelial cells were isolated from kidneys using a combination of collagenase treatment, differential seiving, trypsin treatment and FACS. The RNA was isolated from purified endothelial cells and the gene expression profiles were determined by microarrays.
Project description:The objective of this study is to create an encyclopedia of all genes expressed in the glomerular endothelial cell under normal and diabetic conditions. We utilized Tie2-GFP transgenic mice to mark cells of the glomerular endothelium. To induce diabetic nephropathy (DB), a genetic model of DB, BKS.Cg-m +/+ Leprdb/J from Jax laboratories was used. We utilized fluorescent activated cell sorting (FACS) to isolate glomerular endothelial cells from normal and diabetic mice. The RNAs from these samples were isolated and utilized to hybridize to microarrays, which offers a powerful, efficient and effective method for the creation of a gene expression atlas. Microarrays were used to identify the transciptional differences that occur in the glomular endothelium of a diabetic mouse.
Project description:The pathogenic mechanisms of common kidney glomerular diseases, including the vast majority of cases of proteinuria, remain unknown. To gain insight into the pathogenesis of proteinuria development, we characterized the glomerular gene expression changes that accompany early stages of proteinuria induced by lipopolysaccharide (LPS) in mice.