Abstract
The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-β1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model’s usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models.
Highlights
The glomerulus is the filtration unit of the kidney
GlomSpheres are generated by forming a core of 5000 glomerular endothelial cells (GEnCs), which is wrapped with a peripheral coating of 5000 podocytes (Supplementary Fig. 1a)
Preliminary experiments, whereby spheroids were formed by completely mixing GEnCs and podocytes, showed that cells self-organise over 72 h (Supplementary Fig. 2)
Summary
The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. We report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Utilising more advanced models only for later stages is insufficient, as false positives are compounded at each step of the drug discovery pipeline[3]. GEnCs7 and mesangial cells[8] are the current gold standard in vitro tools and represented a step change in differentiation profile of cultured glomerular cells. These cells are routinely mono-cultured in 2D environments, which (for podocytes at least) has resulted in some degree of phenotypic drift[9]
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