High-content screening assay-based discovery of paullones as novel podocyte-protective agents

Abstract

Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocyte's normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell's filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3β and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte-protective agents for future therapeutic development.

The motor protein Myo1c regulates transforming growth factor-β–signaling and fibrosis in podocytes

“…The control and GDF-15 over-expressing cultured podocytes were treated with protamine sulphate as described previously 77 . Following over-night treatment, the cells were trypsinized and incubated with FITC-annexin V and propidium iodide for 15 min in annexin V binding buffer as described previously 78 . Flow cytometry was performed at the MUSC FACS core facility using BD Fortessa X-20 Analytic Flow Cytometer (BD Biosciences) to detect annexin V-and/or PI-bound cells.…”

Eucalyptol Ameliorates Dysfunction of Actin Cytoskeleton Formation and Focal Adhesion Assembly in Glucose‐Loaded Podocytes and Diabetic Kidney

“…Since GSK3β controls podocyte motility and focal adhesion turnover, eucalyptol therapy might blunt paxillin phosphorylation and boost actin cytoskeleton integrity in podocytes through attenuating GSK3β activation. Paullone derivatives protect podocytes from puromycin aminonucleoside‐mediated injury via suppression of activation of signaling downstream of GSK3β and p38 MAPK . Since GSK3β is a partial downstream mediator of Rho/ROCK signaling in podocyte focal adhesion, eucalyptol may block the link between Rho A and GSK3β activation.…”

Renal cell markers: lighthouses for managing renal diseases

“…However, in the diseased condition, the podocytes are reorganized into a flattened shape with effacement of podocyte FPs and loss of SD sub-structures, culminating in massive proteinuria (>3.5 g albumin/day). Since podocytes are the key cells that are involved not only in glomerular physiology but also in the pathology underlying several nephropathies (15,172,215,348), researchers have studied and identified specific podocyte proteins that have direct association with either the disease outcome or its prognosis and suggested new agents or therapies specific to podocytes (179,259,266).…”