A High-Content Screening Technology for Quantitatively Studying Podocyte Dynamics

Abstract

Podocytes form the visceral layer of a renal glomerulus and express a characteristic octopus—like cellular architecture specialized for the ultrafiltration of blood. The cytoskeletal dynamics and structural elasticity of podocytes rely on the self-organization of highly interconnected actin bundles and the maintenance of these features is important for the intact glomerular filtration. Development of more differentiated podocytes in culture has dramatically increased our understanding of the molecular mechanisms regulating podocyte actin dynamics. Podocytes are damaged in a variety of kidney diseases and therapies targeting podocytes are being investigated with increasing efforts. Association between podocyte damage and disease severity—or between podocyte recovery and the performance of therapeutic molecules—have been the venues of research for years. In this perspective, more standardized high content screening (HCS) has emerged as a powerful tool for visualization and analysis of podocyte morphology. This high-throughput fluorescence microscopy technique is based on an automated image analysis with simultaneous detection of various phenotypes (multiplexing) across multiple phenotypic parameters (multiparametric). Here, we review the principles of HCS technology and summarize efforts to carry out small compound screen using podocytes.

Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

“…Micrographs of structural markers only were adjusted uniformly in Photoshop (Adobe) using the levels function. The confocal micrographs of human kidney biopsies from healthy, early-stage DN, late-stage DN, early-stage FSGS, or late-stage FSGS were analyzed for glomerular expression of ICOSL by manually selecting glomeruli, defined by synaptopodin, as the ysis, the podocytes were imaged using an Opera high-content image system and quantification was performed using Columbus software (Perkin Elmer) (34). To count adhered podocytes, nuclei were detected using DAPI signal, and the nuclei number was counted.…”

Establishing a Cell‐Based High‐Content Screening Assay for TCM Compounds with Anti‐Renal Fibrosis Effects

“…Successful disease-specific tailoring of therapeutics may be achieved by using an image-based screening approach, enabling analyzing a wide variety of cellular phenotypes. Such high-content screening (HCS) platforms employ fully automated microscopes and image analysis software, making it possible to quantify changes in cellular and subcellular properties, including cell area, morphology, actin fibers, and the intensity of focal adhesion [ 8 ]. In various studies, high-content whole-well imaging approaches have been described to evaluate drug activity [ 9 – 11 ].…”

Atypical Renal Clearance of Nanoparticles Larger Than the Kidney Filtration Threshold

“…The final layer is composed of podocytes: octopus-shaped cells which wrap around the glomeruli [ 61 ]. The cells possess “foot processes”, which intercalate with other podocytes, forming “slits” [ 62 ].…”