title: A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules.
abstract: Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an β1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active β1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active β1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Ha Won
authors: HW
authors: Lee
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Samia Q
authors: SQ
authors: Khan
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Mohd Hafeez
authors: MH
authors: Faridi
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Changli
authors: C
authors: Wei
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Nicholas J
authors: NJ
authors: Tardi
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Mehmet M
authors: MM
authors: Altintas
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Hatem A
authors: HA
authors: Elshabrawy
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois;
authors: Steve
authors: S
authors: Mangos
authors: Cellular Imaging and Analysis, PerkinElmer Life Sciences, Waltham, Massachusetts; and.
authors: Kevin L
authors: KL
authors: Quick
authors: Department of Medicine, Harvard Medical School, Division of Nephrology, Massachusetts General Hospital, Charlestown, Massachusetts.
authors: Sanja
authors: S
authors: Sever
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois; jochen_reiser@rush.edu vineet_gupta@rush.edu.
authors: Jochen
authors: J
authors: Reiser
authors: Department of Medicine, Rush University Medical Center, Chicago, Illinois; jochen_reiser@rush.edu vineet_gupta@rush.edu.
authors: Vineet
authors: V
authors: Gupta
mesh_terms: D000199:Actins / Q000378:metabolism; D000419:Albuminuria / Q000378:metabolism; D000818:Animals; D002465:Cell Movement; D004847:Epithelial Cells / Q000187:drug effects; D022001:Focal Adhesions / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006912:Hydroxyquinolines / Q000737:chemistry*; D019012:Integrin beta1 / Q000378:metabolism*; D007674:Kidney Diseases / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D010641:Phenotype; D050199:Podocytes / Q000166:cytology*; D011507:Proteinuria / Q000473:pathology; D011692:Puromycin Aminonucleoside / Q000737:chemistry; D051381:Rats; D013449:Sulfonamides / Q000737:chemistry*
chemical_list: D000199:Actins; D006912:Hydroxyquinolines; D019012:Integrin beta1; D008070:Lipopolysaccharides; D013449:Sulfonamides; C000605137:pyrintegrin; D011692:Puromycin Aminonucleoside
keywords: adhesion molecule; glomerular disease; glomerular epithelial cells