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

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.


Supplementary Figures
. TFCH displays better sensitivity to cellular carbonylated biomolecules than CH. A549 lung cancer cells grown in standard media (A) or serum free media (SFM) (B) were incubated with 20 µM CH or TFCH for 2.5 h. The cells were then washed, lysed, and an emission spectrum of each sample was recorded (excited at 405 nm). The spectra were normalized by total protein representing total cell number. The relative fluorescence of each sample is reported as the total area under the curve (AUC). Note that the fluorescence in the control samples is due to the presence of inherent carbonyls in cancer cells 1 .

Figure S6. TFCH serves as a tool for detecting oxidative stress-induced carbonylation in live cells by HCS-and HTS-compatible assays.
A plate-reader based assay for detecting SFM-induced carbonylation (A-B). A549 cells were grown in standard media (control) or SFM for 24 h before adding the stated concentration of TFCH for 90 min. The cells were rinsed with PBS and the emission was recorded at 525 nm (excitation: 405 nm). Graph shows relative fluorescence due to serum starvation detected by different concentrations of TFCH (A). Error bars represent SEM. The fold increase in fluorescence was calculated as the ratio of signal generated in the SFM treated samples and the control samples (B). A one-step assay for visualizing biomolecule-carbonyls in live cells (C). Serum-starved or control cells were allowed to react with 2 µM TFCH for 30 min before imaging the live cells without rinsing out excess fluorophore. Scale bar, 20 μm. A two-step assay for visualizing biomolecule-carbonyls (D-E). A549 cells grown in standard media (control) or SFM for 24 h were allowed to react with 20 µM TFCH for 30-60 min. In the secondstep, excess fluorophore was removed; the cells were washed and either imaged live (D) or fixed, processed (as described in the Methods), and imaged (E). Scale bar, 20 μm.

Figure S7. TFCH is a live cell compatible probe for detecting OS-induced carbonylation in kidney cells.
TFCH is not cytotoxic (A-B). Resazurin (A) or SRB (B) assay was performed after MDCK cells were exposed to media without added treatment (control), with vehicle (0.5%, v/v, DMSO) or with TFCH (20 µM, the highest concentration used) for 24 h. Error bars represent SD. One-way ANOVA with Dunnett's multiple comparisons test was performed to compare each treatment with the control; P>0.05 was considered not significant (ns).

TFCH detects oxidative stress-induced carbonylation in kidney cells (C-E)
. SFM (C)-, H2O2 (D)-or menadione (E)-induced carbonylation was detected by incubating live LLC-PK1 or MDCK cells with 20 µM TFCH for 30-60 min. Cells were rinsed with PBS, fixed, and processed before imaging. Scale bar, 20 µm. Bar graphs showing quantification of cellular carbonyls detected by TFCH in LLC-PK1 or MDCK cells. At least three independent experiments were performed, and fluorescence associated with > 100 cells were quantified. An unpaired t-test with Welch's correction was performed. ****P <0.0001. Error bars represent SEM.  . TFCH (20 µM) was added to the medium for an additional 60 min. The cells were then rinsed with PBS, fixed, processed, and imaged as described in the Methods. One-way ANOVA with Dunnett's multiple comparisons test was performed to compare each treatment with the control. ****P <0.0001. Error bars represent SEM. Scale bar, 20 µm. Quantification of renal tubule associated fluorescence in control (no drug), cisplatin (150 µg/mL), or gentamicin (4.6 mg/mL) treated tissue slices (C). Error bars represent SD. Control samples (D): tissue sections treated with cisplatin (150 µg/mL) or gentamicin (4.6 mg/mL) were exposed to vehicle (0.5% DMSO, v/v), instead of TFCH. An unpaired t-test with Welch's correction was performed to compare the control with each treatment. ****P <0.0001. Error bars represent SD. Scale bar, 100 µm.
Stannous chloride dihydrate (1.57 mg, 3.8 eq) was dissolved in 1.50 mL concentrated HCl and chilled on ice. This cold stannous chloride HCl solution was then slowly added to the diazonium solution and the temperature of reaction was maintained below 0 ºC. The reaction was stirred for 1.5 h at -10 ºC. The yellow slurry was then filtered and washed with cold water and cold ethanol. TFCH HCl salt was collected as a light yellow solid (350 mg, 57% yield). The TFCH HCl salt (20 mg, 0.082 mmol) was dissolved in 600 μL methanol and 20 μL of trifluoroacetic acid. Propionaldehyde (58.7 μL, 10 eq) was added to the solution which was stirred further for 30 min. The yellow precipitate was filtered and washed with cold methanol. The TFCZ was dried by air and collected as yellow solid (15 mg 64% yield).

TFCH reaction with propanal.
Equal volumes of 10 µM TFCH and 1 mM propanal in 10 mM PB, pH 7 containing 0.5% DMSO placed in separate compartments of a dual chambered quartz cell in an absorption spectrophotometer (Hewlett-Packard 8453). The sample was zeroed and the solution was quickly mixed by inversion. Spectra were obtained over time until a steady state was reached. Note, the solutions are diluted by a factor of two and the path length is doubled; therefore, only the differences in the absorbance of the solution after mixing are recorded.
Hydrazone formation between 5 µM TFCH and 500 µM propanal in 10 mM PB, pH 7 containing 0.5% DMSO was monitored using Synergy Mx microplate reader (BioTek). The sample was excited at 388 nm and the emission at 550 nm was recorded over time until a steady state was reached.
Absorption spectra of these samples were taken at room temperature (RT) using a Hewlett-Packard 8452A diode array spectrophotometer equipped with the Olis Globalworks software. Emission and excitation spectra were recorded at RT using a Jobin Yvon Horiba FluoroMax-3 spectrofluorometer. Fluorescence spectra were collected using a 2 × 10 mm fluorescence cuvette, positioned for the light to pass through the shorter path. Emission spectra (405 nm excitation) were corrected by subtracting the solvent's fluorescence followed by normalization to account for any differential absorption of TFCH and TFCZ at the excitation wavelength.

Detection of carbonylated BSA by TFCH
Bovine serum albumin (BSA) was oxidized as described previously 2 . Commercially available unmodified BSA or oxidized BSA was allowed to react with TFCH for 3.5 h. In parallel, oxidized BSA was allowed to react with TFCA for the same period of time. The final concentrations of the protein and the fluorophore in each reaction were 2 µg/µL and 300 µM, respectively. The samples were then subjected to SDS-PAGE analysis. The gel was imaged under long wavelength UV prior to Coomassie blue staining and imaging to account for protein loading. The major band representing BSA is shown in the illustration.
Cell culture A549 cells were grown in F12K media (ATCC); MDCK cells were grown in DMEM:F-12 (ATCC); and LLC-PK1 cells were grown in DMEM with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning).
All standard cell culture media were supplemented with 10% FBS (ThermoFisher Scientific, Sigma or Atlanta Biologicals) and 1% antibiotic-antimycotic (ThermoFisher Scientific). All serum-free media (SFM) were supplemented only with 1% antibiotic-antimycotic. Cells were maintained in a mammalian cell culture incubator. LLC-PK1 cells were grown for 72 h and MDCK cells were grown overnight on collagen-coated coverglasses or 96-well plates prior to treatment.

SFM-induced
Control (cells grown in standard media) or serum-starved (cells grown in SFM) A549 cells were incubated with 2 µM TFCH in 0.5% DMSO for 30 min and imaged immediately, without washing out excess fluorophore 3 .
In another set, A549 cells were grown in standard media or SFM for 24 h followed by incubation with 20 µM TFCH in 0.5% DMSO for 30 min. The cells were washed and imaged live at room temperature within 20 min after fluorophore incubation using a Zeiss confocal microscope. Ex: 405 nm; Em: LP 420. In an independent experimental set, MDCK cells were incubated with standard media (control) or SFM for 1.5 h and then allowed to react with 20 µM CH, BzCH, DCCH or TFCH for 30 min. The cells were rinsed, fixed, processed, and imaged as described previously. CH used in this work was either purchased from Cayman Chemicals or synthesized in the Bane lab 2 , BzCH was synthesized in the Bane lab 4 , and DCCH was purchased from Sigma.
The media was replaced with fresh media containing 20 µM TFCH in 0.5% DMSO (v/v). After a 30 min incubation, the cells were washed, fixed, processed, and imaged as described previously.

Menadione-induced
MDCK or LLC-PK1 cells were treated with vehicle (DMSO, 0.05% v/v) or 100 µM menadione for 1 h prior to the addition of 20 µM TFCH in 0.5% DMSO (v/v). After 30 min, the cells were washed, fixed, mounted, and imaged as described previously.

Gentamicin and Cisplatin-induced
LLC-PK1 or MDCK cells grown on collagen-coated coverglasses were treated with 0.15 μg/mL of cisplatin (cis-Diamineplatinum(II) dichloride, Sigma) or 0.58 mg/mL of gentamicin (gentamicin sulfate salt, Sigma) for 24 h. A final concentration of 20 µM TFCH in 0.5% (v/v) DMSO was added to the samples and incubated for 40-60 min before washing, fixing, processing, and imaging. Cells that did not receive any drug treatment were used as a control.
In another experimental set, LLC-PK1 or MDCK cells were allowed to grow for 3 h in the presence of stated concentrations of cisplatin (Cis) or gentamicin (Gen). TFCH (20 µM) was then added to the medium for an additional 60 min, washed, fixed, and processed before imaging.
CellROX Green-mediated detection of reactive oxygen species (ROS) in different models of oxidative stress All treatment methods employed for preparing the samples for subsequent staining with CellROX Green (ThermoFisher Scientific) were the same as that of TFCH. The CellROX Green staining was performed according to the manufacturer's instructions. Briefly, the cells were treated with 5 μM CellROX Green for 30 min, washed, fixed, processed (as described previously) before imaging.

Image acquisition, quantification and analysis for cell-based assays using TFCH and CellROX Green
Cells stained with TFCH were excited using the 405 nm laser and the emission was monitored using LP420 or between 420-700 nm. Cells stained with CellROX Green were excited using the 488 nm laser and emission was monitored using LP505.
All images were obtained using a Zeiss or a Leica confocal microscope. For visual clarity, the photomicrographs were processed using identical parameters for each cell line within an experimental set.
Thresholded photomicrographs were used for quantification of signal/cell (IntDen/cell) using Fiji/ImageJ (NIH). Data are reported as mean ± SEM of signal per cell relative to the average of the control. The average of the control in each experimental set was set to 100. Data from a minimum of 2 experimental sets, with a minimum of 30 cells per sample per set, are reported in the graph. Where indicated, a two-tailed unpaired t-test with Welch's correction or a one-way ANOVA with Dunnett's multiple comparisons test was performed using GraphPad Prism to compare each treatment with the control. The percent difference in mean and standard error was also calculated using GraphPad Prism. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and not significant (ns) was p>0.05.

Resazurin-based assay
Cisplatin or gentamicin drug stocks were prepared fresh in appropriate cell culture media prior to each experiment. LLC-PK1 or MDCK cells grown on collagen-coated 96-well plates (100 μL media/well) were treated with only media (control), cisplatin or gentamicin by adding an additional 100 μL of fresh media supplemented with or without drugs. In parallel, a separate set of wells that did not contain cells were similarly treated for background subtraction. After 20-22 h, the cell viability assay was initiated using the In vitro toxicology assay kit, Resazurin based (Sigma-Aldrich) following the manufacturer's instruction. In brief, 22 μL of the reagent was added to each well and incubated for 4 h. The samples were excited at 560 nm and emission was recorded at 590 nm using a Cytation 5 imaging reader (BioTek). Emission of the treated wells without cells was subtracted from the emission of those containing cells. Each experimental set consisted of control cells (no drug treatment), and treatment with cisplatin or gentamicin. The graph represents the effect of drugs on cell viability relative to the control (without drug treatment). The average of the control in each experimental set was set to 100%. Error bars show SEM. Where indicated, an unpaired t-test with Welch's correction was performed and the percent difference in mean and standard error was calculated using GraphPad Prism.

SRB-based assay
The drug treatment was performed as described in the resazurin-based assay section. After 24 h incubation with different drug concentrations, a SRB-based assay was performed as described before with minor modifications 2 . Experimental layout and data presentation are the same as that of the resazurin-based assay.
Where indicated, an unpaired t-test with Welch's correction was performed and the percent difference in mean and standard error was calculated using GraphPad Prism.

Effect of TFCH on cell viability
MDCK cells were treated with only media (control), vehicle (0.5%, v/v, DMSO) or TFCH (20 µM, the highest concentration used) for 24 h before performing a resazurin-or SRB-based assay. One-way ANOVA with Dunnett's multiple comparisons test was performed to compare each treatment with the control using GraphPad Prism.
Actin and ZO-1 LLC-PK1 and MDCK cells grown on collagen-coated coverglasses were treated with different concentrations of drugs. After 24 h, the samples were processed for immunocytochemistry to assess the status of ZO-1 and actin. A polyclonal ZO-1 antibody (ThermoFisher Scientific), followed by AlexaFlour 488 goat anti-rabbit IgG (H+L), was used to stain ZO-1. Subsequently, the cells were stained with Rhodamine Phalloidin and Hoechst 33342 to probe for F-actin and the nucleus, respectively. Cells were imaged using a Zeiss confocal microscope. The intensity of actin-stress fibers within each cell (IntDen/cell) was quantified by Fiji/ImageJ (NIH) in thresholded images. The intensity (IntDen) of cell membraneassociated ZO-1 was quantified by selecting a fixed region of interest drawn with the "rectangle" tool.
Emission channels representing actin and ZO-1 staining are presented in the figure. For visual clarity, the photomicrographs were processed using identical parameters for each cell line within an experimental set.
Graphs represent the intensity of actin stress fiber per cell or ZO-1 intensity, both relative to the control.
The average of the control was set to 100. An unpaired t-test with Welch's correction was performed and the percent difference in mean and standard error was calculated using GraphPad Prism.