Abstract
BackgroundEndothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.ResultsFlow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.ConclusionIn this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0040-6) contains supplementary material, which is available to authorized users.
Highlights
Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis
Endothelial cells have been found to be a major source of myofibroblasts via endothelial mesenchymal transition (EndoMT), causing fibrosis in kidney and other organs [1,2,3,4]
Zeisberg and colleagues [4] showed in three mouse models including unilateral ureteral obstructive nephropathy (UUO), Alport disease and streptozotocin-induced diabetic nephropathy, that around 30% to 50% of fibroblasts formed in the kidneys coexpressed the endothelial marker CD31 and the fibroblast/ myofibroblast markers fibroblast specific protein-1 (FSP-1) and/or α-smooth muscle actin (α-SMA)
Summary
Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. Successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. Endothelial cells have been found to be a major source of myofibroblasts via endothelial mesenchymal transition (EndoMT), causing fibrosis in kidney and other organs [1,2,3,4]. Zeisberg and colleagues [4] showed in three mouse models including unilateral ureteral obstructive nephropathy (UUO), Alport disease and streptozotocin-induced diabetic nephropathy, that around 30% to 50% of fibroblasts formed in the kidneys coexpressed the endothelial marker CD31 and the fibroblast/ myofibroblast markers fibroblast specific protein-1 (FSP-1) and/or α-smooth muscle actin (α-SMA). By immunofluorescence staining of kidney sections of mice with UUO, co-localization of the mesenchymal marker α-SMA and endothelial marker CD31 or VE-
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