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 Z9 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 b1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active b1-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 b1 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.

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1

“…Actin cytoskeleton in podocytes is a critical link between various signaling components and the cellular receptors, whereas playing an essential role in providing stable architectural support to the cell. Immunofluorescence microscopy showed that TGF-␤1 induced rearrangement of the actin cytoskeleton resulting in loss of stress fibers in podocytes (58,59), a hallmark of cellular damage, and that this loss of stress fibers was prevented by co-treatment with erlotinib ( Fig. 8A), suggesting a link between TGF-␤1 signaling and ErbB4.…”

“…After incubating with primary antibodies, secondary antibodies and/or Alexa 568-conjugated phalloidin (Life Technologies) were incubated with cells. Stained cells were imaged with a Zeiss 700 LSM confocal microscope (Zeiss, Hartford, CT) and Opera Imaging System (PerkinElmer Life Sciences), as described (58).…”

“…Briefly, kidney sections from 25-week-old miR-146a Ϫ/Ϫ mice treated with vehicle (sodium citrate), STZ, or STZ and erlotinib were sectioned and imaged as recently described (58). Images were acquired using Zeiss Sigma HD VP electron microscope (Zeiss).…”

Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling

“…Consistent with our hypothesis, we observed that cilengitide blocked all of the effects of Sdc4 ectodomain that we have observed in podocytes, including the increased surface expression of TRPC6 channels, increased ROS generation, modulation of small GTPases, and activation of NFATc1. In this regard, several antagonists of outside-in integrin signaling have protective effects in animal models of podocyte diseases, including diabetic nephropathy in pigs [49] and rats [50], lipopolysaccharide-induced podocyte effacement [51], acute PAN nephrosis [51], and the early stages of experimental glomerulonephritis [52]. There is also evidence that constitutive activation of α v β3 integrins in podocytes leads to albuminuria, glomerulosclerosis, and activation of Rac1 [53].…”

Targeting the podocyte to treat glomerular kidney disease

“…Although there are important limitations to consider when utilizing such podocyte cell lines, podocyte cell culture has been an instrumental tool for simulating various aspects of glomerular disease biology. From the drug discovery perspective, there remain significant opportunities to take advantage of this cellular system for target evaluation and discovery [13,35].…”