A Novel Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue

Abstract

Drug-induced kidney injury frequently leads to aborted clinical trials and drug withdrawals. Sufficiently sensitive sensors capable of detecting mild signs of chemical insult in cell-based screening assays are critical to identifying and eliminating potential toxins in the preclinical stage. Oxidative stress is a common early manifestation of chemical toxicity, and biomolecule carbonylation is an irreversible repercussion of oxidative stress. Here, we present a novel fluorogenic assay using a sensor, TFCH, that responds to biomolecule carbonylation and efficiently detects modest forms of renal injury with much greater sensitivity than standard assays for nephrotoxins. We demonstrate that this sensor can be deployed in live kidney cells and in renal tissue. Our robust assay may help inform preclinical decisions to recall unsafe drug candidates. The application of this sensor in identifying and analyzing diverse pathologies is envisioned.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury

“…As altered actin dynamics can further enhance ROS production, we next examined whether stabilization of the actin network by Bis-T-23 can counteract oxidative stress in cisplatin-injured cells. We elected to assess the status of cellular biomolecule carbonyls, a stable biomarker of oxidative damage produced by high levels of ROS, using our recently developed fluorescent sensor TFCH 37 (Fig. 4e ).…”

“…Mitochondria are dynamic organelles that respond to physiological signals and are significant sources of ROS in healthy 60 and injured cells 61 . Cisplatin and iohexol directly damage mitochondria leading to increased production of ROS 37 , 43 , 62 – 64 , while suPAR affects the bioenergetic parameters of renal cells 47 . As many cytoskeletal proteins are sensitive to ROS 32 , 65 , a decrease in actin dynamics in the presence of elevated levels of ROS has been reported 32 .…”

“…Bis-T-23 (Ryngo 1-23), Dynole 34-2 (Abcam); Latrunculin A (LatA), cytochalasin D (CytoD), jasplakinolide (Jasp), rhodamine-phalloidin, rhodamine-transferrin, rhodamine-albumin, Hoechst 33342, ProLong TM Gold antifade reagent (ThermoFisher Scientific). Glutathione Sepharose 4B (GE Healthcare); protein G PLUS-agarose beads (Santa Cruz Biotechnology); TFCH (Bane laboratory, Binghamton University, SUNY) 37 ; cisplatin, NG-nitro-L-arginine methyl ester (L-NAME), indomethacin (Sigma-Aldrich); iohexol (Omnipaque, 350 mg iodine/ml, TCI America). For cell-based assays, cisplatin and iohexol were dissolved in media before use; other small molecule solutions were prepared in 100% DMSO and frozen until use.…”

“…MDCK cells were treated with DMSO (0.1%, vehicle for Bis-T-23 or LatA) or Bis-T-23 (10 µM) for 20 min before adding DMSO or LatA (0.2 µM) for 10 min. TFCH (20 µM, 0.5% DMSO) was added for 30 min, and samples were processed for imaging 37 .…”

Drug-Induced Nephrotoxicity Assessment in 3D Cellular Models

“…Even with the advancement of medical technology, the incidence of AKI has gradually increased in recent years, leading to an increase in patient mortality [4]. A variety of risk factors may contribute to the occurrence of AKI, including food preparations, drugs, infection, ischemia, sepsis, and intravenous contrast agents [5,6]. In particular, drug-induced nephrotoxicity is a major contributing factor in approximately 60% of AKI cases in hospitalized patients [7].…”