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Drugs Repurposed as Antiferroptosis Agents Suppress Organ Damage, Including AKI, by Functioning as Lipid Peroxyl Radical Scavengers

Eikan Mishima ,1 Emiko Sato,1,2 Junya Ito,3 Ken-ichi Yamada,4 Chitose Suzuki,1 Yoshitsugu Oikawa,5 Tetsuro Matsuhashi,5 Koichi Kikuchi,1 Takafumi Toyohara,1 Takehiro Suzuki,1 Sadayoshi Ito,1,6 Kiyotaka Nakagawa,3 and Takaaki Abe 1,7,8

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT Background Ferroptosis, nonapoptotic cell death mediated by free radical reactions and driven by the oxidative degradation of lipids, is a therapeutic target because of its role in organ damage, including AKI. Ferroptosis-causing radicals that are targeted by ferroptosis suppressors have not been unequivocally iden- tified. Because certain cytochrome P450 substrate drugs can prevent lipid peroxidation via obscure mecha- nisms, we evaluated their antiferroptotic potential and used them to identify ferroptosis-causing radicals. Methods Using a cell-based assay, we screened cytochrome P450 substrate compounds to identify drugs with antiferroptotic activity and investigated the underlying mechanism. To evaluate radical-scavenging activity, we used electron paramagnetic resonance–spin trapping methods and a fluorescence probe for lipid radicals, NBD-Pen, that we had developed. We then assessed the therapeutic potency of these drugs in mouse models of cisplatin-induced AKI and LPS/galactosamine-induced liver injury. Results We identified various US Food and Drug Administration–approved drugs and hormones that have antiferroptotic properties, including rifampicin, promethazine, omeprazole, indole-3-carbinol, carvedilol, pro- pranolol, estradiol, and thyroid hormones. The antiferroptotic drug effects were closely associated with the scavenging of lipid peroxyl radicals but not significantly related to interactions with other radicals. The ele- vated lipid peroxyl radical levels were associated with ferroptosis onset, and known ferroptosis suppressors, such as ferrostatin-1, also functioned as lipid peroxyl radical scavengers. The drugs exerted antiferroptotic activities in various cell types, including tubules, podocytes, and renal fibroblasts. Moreover, in mice, the drugs ameliorated AKI and liver injury, with suppression of tissue lipid peroxidation and decreased cell death. Conclusions Although elevated lipid peroxyl radical levels can trigger ferroptosis onset, some drugs that scavenge lipid peroxyl radicals can help control ferroptosis-related disorders, including AKI.

JASN 31: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2019060570

Ferroptosis is an iron-dependent nonapoptotic reg- reported.4–6 Among renal disorders and diseases, ulated cell death characterized by extensive lipid ferroptosis has been reported to contribute to the peroxide accumulation, which is induced by dis- rupting the glutathione-dependent lipid peroxide defense system.1,2 Emerging evidence implicates Received June 6, 2019. Accepted October 17, 2019. ferroptosis in various disorders, including acute or- Published online ahead of print. Publication date available at gan injury and neurodegenerative diseases, www.jasn.org. and in a cellular mechanism that promotes tumor Correspondence: Dr. Eikan Mishima, Division of Nephrology, suppression.3 The pathophysiologic importance of Endocrinology and Vascular Medicine, Tohoku University, Sendai, ferroptosis in stroke, myocardial infarction, and Miyagi, Japan. Email: [email protected] post-transplant immune response has also been Copyright © 2019 by the American Society of Nephrology

JASN 31: ccc–ccc,2019 ISSN : 1046-6673/3102-ccc 1 BASIC RESEARCH www.jasn.org pathophysiology in AKI, ischemia-reperfusion damage, dru- Significance Statement g-induced kidney injury, rhabdomyolysis-associated renal damage, and growth of renal cysts.7–11 Thus, a better under- Ferroptosis, cell death mediated by free radical reactions and standing of ferroptosis and the development of an effective driven by oxidative degradation of lipids, is a therapeutic target intervention could lead to therapies for these disorders. because of its role in organ injuries, including AKI. However, the ferroptosis-causing radicals targeted by ferroptosis suppressors Ferroptosis onset can be prevented by lipid peroxidation have not been unequivocally identified. Certain cytochrome P450 suppression, indicating that this form of cell death is pharma- substrate drugs are known to prevent lipid peroxidation via obscure cologically accessible. Because lipid peroxidation is mediated mechanisms. The authors screened cytochrome P450 substrate by nonenzymatic radical reactions or enzymatic lipoxygenase drugs, identifying a diverse group of drugs with antiferroptotic (LOX) activity in the presence of iron,12 radical-trapping properties, including promethazine and rifampicin. The anti- ferroptotic effect of these drugs was linked to their scavenging agents, LOX inhibitors, and iron chelators prevent ferroptosis activity against lipid peroxyl radicals. Elevated lipid peroxyl radical by blocking lipid peroxidation.3 In addition to known ferrop- levels were associated with ferroptosis onset, whereas radical tosis suppressors such as the lipophilic antioxidants ferrosta- scavenging by the drugs suppressed ferroptosis-related pathologic tin-1 (Fer-1) and liproxstatin-1, several other antioxidant changes in different renal cell types and ameliorated organ injuries compounds have been reported to exert antiferroptotic ef- (including AKI) in mice, suggesting therapeutic potential for such repurposed drugs. fects,13–15 suggesting the involvement of reactive oxygen species (ROS) in ferroptosis. However, the key radical species that are targeted by ferroptosis suppressors have not been mechanisms are not well understood.25–27 We hypothesized unequivocally identified. that these CYP substrate types might prevent ferroptosis by Lipid peroxidation is propagated by a chain reaction of lipid blocking lipid peroxidation and proposed that the elucidation peroxyl radicals.16 During the nonenzymatic step of lipid per- of the antiferroptotic mechanisms of these drugs may help to oxidation initiation, a lipid radical reacts with oxygen to form determine the causative radicals and activities associated with the lipid peroxyl radical, which can generate a lipid hydroper- ferroptosis onset. In this study, we tested CYP substrates to oxide and another lipid radical, leading to a chain reaction. identify drugs with antiferroptotic properties. To investigate In the LOX pathway, a polyunsaturated fatty acid is converted the mechanism, utilizing NBD-Pen and the ESR–spin trapping into lipid hydroperoxide, which is decomposed to a lipid per- method, we examined the radical-scavenging activity of the oxyl radical to initiate the chain reaction. Thus, scavenging potential antiferroptotic drugs especially toward lipid peroxyl lipid peroxyl radicals could stop the radical chain reaction, radicals. In addition, we evaluated the therapeutic potency of which would prevent ferroptosis. It has been proposed the drugs in organ injury models of mice including AKI. Our that the mechanism of the antiferroptotic effect of the radi- findings suggested that the elevated lipid peroxyl radical levels cal-trapping agents is on the basis of their scavenging activity were associated with ferroptosis onset, whereas scavenging toward lipid ROS.17 However, evidence for their lipid ROS lipid peroxyl radicals by the drugs suppressed ferroptosis- scavenger activity has been insufficient because of the techni- related pathologic conditions. cal difficulties associated with performing direct measure- ments of lipid radicals. Although antioxidative activities of Fer-1 and liproxstatin-1 have been evaluated by standard METHODS assays measuring 2,2-diphenyl-1-picrylhydrazyl reduction ac- tivity and suppression of C11-BODIPY581/591 oxidation,14,18 Detailed methods are shown in the Supplemental Material. these methods did not directly demonstrate scavenging ac- tivity toward lipid radicals. The only currently established Cell Lines method to monitor lipid radicals uses electron spin reso- H9C2 (rat cardiomyoblast), NRK49F (rat kidney fibroblast), nance (ESR)–spin trapping techniques,19 but we recently HK2 (human kidney tubular cell), C2C12 (mouse myoblast), developed a specific fluorescence probe for lipid radicals, MDA-MB-231 (human breast adenocarcinoma), NRK52E 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)- (rat kidney tubular cell), and LLC-PK1 (porcine kidney tubu- 6-pentylpiperidine-1-oxyl (NBD-Pen), which enabled us to lar cell) cells were obtained from ATCC. Panc-1 (human pan- directly detect lipid-derived radicals with high sensitivity and creas carcinoma) cells were obtained from the Cell Resource selectivity.20 Center for Biomedical Research, Tohoku University (Sendai, Cytochrome P450 families (CYP) contain key enzymes of Japan). HT-22 cells were purchased from Millipore. Human drug metabolism and steroid hormone synthesis that can also urine-derived podocyte-like epithelial cells (HUPECs) were convert lipophilic substrates to hydrophilic products.21 Some provided by Dr. Jeffrey B. Kopp (National Institutes of Health, CYP-catalyzed reactions are involved in ROS production and Bethesda, MD).28 AllcelllinesexceptHUPECweremain- lipid peroxidation.12,22 Earlier in vitro studies on microsomes tained in DMEM high glucose (4.5 g glucose/L) supplemented demonstrated that certain CYP substrates and CYP-inducing with 10% FBS, and 1% penicillin/streptomycin at 37°C under 23,24 compounds inhibit lipid peroxidation, and some of these 5% CO2. HUPEC was maintained in RPMI 1640 supplemen- substrates have cytoprotective effects, but the involved ted with 10% FBS and 1% insulin, transferrin, and selenium

2 JASN JASN 31: ccc–ccc,2019 www.jasn.org BASIC RESEARCH solution (Thermo Scientific) at 33°C and incubated at 37°C previously.30,31 The scavenging potential of the compounds during the experiments. to 2,29-azobis(2-amidinopropane) (AAPH)–derived peroxyl radical was assessed using OxiSelect ORAC Activity Assay Screening of Antiferroptotic Compounds Kit (Cell Biolabs). H9C2 cells plated in 96-well plates with DMEM (1.0 g glucose/L, 1% FBS) were cotreated with 100 mM L-buthionine sulfoxi- Lipid Peroxyl Radical–Scavenging Analysis mine (BSO) and a dilution series of compounds listed in Sup- Lipid peroxyl radicals generated by arachidonic acids (AAs)/ plemental Table 1. After incubating for 60 hours, cell viability LOX system were detected with the NBD-Pen fluorescence was measured using Cell Count Reagent SF (Nacalai tesque) probe.20 Mixtures of 160 ml PBS pH 7.4 (PBS) containing AAs and expressed as relative values in relation to that measured (final 500 mM; Sigma-Aldrich [.95.0%]) and the compounds in non-BSO–treated samples defined as 1.0. When the mean were prepared in a black-walled 96-well plate. Aliquots of 20 ml relative value of cell viability was .0.5 at any of the screened NBD-Pen (final 5 mM) and 20 ml LOX solution (final 10 mg/ml, compound concentrations, the respective compound was LOX from Glycine max; Sigma-Aldrich) were added, and the considered having antiferroptotic activity. fluorescence intensity (ex 470 nm, em 530 nm) was measured at 37°C using a Spectra Max M2e (Molecular Devices). Induction of Ferroptosis and Cell Death In BSO experiments, BSO and the compounds were added Phosphatidylcholine Hydroperoxide Experiments to the culture medium simultaneously, and the cell viability Phosphatidylcholine hydroperoxide (PCOOH) was enzymat- andLDHreleasewerethenmeasuredattheindicatedtime. ically synthesized from 16:0–18:2 PC (Avanti Polar Lipids) and In the other cell death experiments, compounds were added chromatographically purified.32,33 For in vitro detection of to the culture medium 1 hour before treatment with cell PCOOH-derived peroxyl radical, mixtures of 190 ml PBS con- death inducers. Released LDH was evaluated using the taining indicated concentrations of PCOOH and NBD-Pen LDH Cytotoxicity Detection kit (Takara Bio). (final 5 mM) were prepared. Aliquots of 10 ml Fe(NH4)2(SO4)2 (final 50 mM) or water were added to the mixtures, and the Evaluation of Mitochondria, Malondialdehyde, Lipid fluorescence intensity (ex 470 nm, em 530 nm) was measured. Hydroperoxides, Glutathione, LOX Inhibitory Ability, To visualize cellular lipid-derived radicals, cells were stained and Iron-Chelating Capacity with NBD-Pen. Ten minutes after replacing culture medium Mitotracker Green FM (Thermo Scientific) and dihydrorhod- with phenol red–free DMEM containing 1% FBS and NBD- amine 123 (Wako) were used for the evaluation of mitochon- Pen (1 mM), cells were rinsed twice with PBS and then in- drial morphology and mitochondrial ROS, respectively, in cubated with phenol red–free DMEM with 1% FBS with or H9C2 cells. Malondialdehyde (MDA) in the medium was without PCOOH for 1.5 hour at 37°C. Fluorescence imaging measured using the TBARS Fluorometric Microplate Assay was conducted with a BZ-X800 fluorescence microscope (Oxford Biochemical Research). Cellular lipid hydroperoxides (Keyence) and GFP filter cube. were detected using the Liperfluo (Dojindo). The GSH-Glo Glutathione Assay (Promega) was used to quantify total glu- Animal Experiments tathione in cultured cells. The determination of LOX inhibi- Animal experiments were performed according to the appli- tory ability was performed using a LOX inhibitor screening kit cable national guidelines for the care and use of laboratory (Cayman). Ferrous oxidation/xylenol orange (FOX) assay was animals and approved by the Animal Committee of Tohoku performed as described previously.29 University, School of Medicine (approval Nos 2016–008–5, 2016–009–2, 2017–014–1, and 2019BeA-012). C57BL/6N siRNA and Generation of Aryl Hydrocarbon Receptor– male mice (CLEA Japan), aged 8–9weeks,wereused.AKI Deficient Cells was induced by intraperitoneal injection of cisplatin solution For the knockdown of Adrb1 expression, Silencer Select Pre- (16 or 17 mg/kg as indicated; Nichi-Iko Pharmaceutical).34 designed siRNA (s236469; Thermo Scientific) and Lipofect- Mice were orally treated with water only, promethazine amine RNAiMAX (Thermo Scientific) were used. Aryl (20 mg/kg in water), or rifampicin (20 mg/kg in 0.5% meth- hydrocarbon receptor (Ahr)–deficient H9C2 cells were gener- ylcellulose) every 12 hours for 4 days starting 30 minutes ated using the sgRNA CRISPR/Cas9 All-In-One kit before the cisplatin injection, or orally treated with prometha- (K6920005; Applied Biologic Materials). For quantitative zine (20 mg/kg) in the following groups: (1) no promethazine, PCR, TaqMan probes (Thermo Scientific) were used: (2) pretreatment 30 minutes before cisplatin injection, (3) Cyp1a1 (Rn00487218_m1) and Actb (Rn00667869_m1). treatment from 30 minutes before injection to 24 hours after injection, (4) treatment from 24 to 96 hours after injection, and Free Radicals–Trapping Analysis (5) treatment every 12 hours from 30 minutes before injection Scavenging potentials of the compounds to superoxide and to 96 hours after injection. They were euthanized 4 days after hydroxyl radical were assessed by ESR–spin trapping using the injection. Acute liver injury was induced by intraperito- 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as described neal injection of Lipopolysaccharide (LPS, 5 mg/kg) and

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 3 BASIC RESEARCH www.jasn.org

D-galactosamine N (GalN; 500 mg/kg) dissolved in PBS.35 indole-3-carbinol, rifampicin, promethazine, carvedilol, pro- Mice were orally treated with water or promethazine pranolol, estradiol, and triiodothyronine (T3). According to (20 mg/kg) 12 hours and 2 hours before the injection and cell viability counts, cytotoxicity assays, and cell morphology euthanized 6 hours after the injection. At the end of each assessments, all positive candidates completely prevented study,themicewereeuthanizedbyisoflurane, and samples BSO-induced cell death despite the diversity of their reported were collected. pharmacologic activities (Figure 1C). These drugs also pre- vented ferroptotic cell death induced by other ferroptosis in- Histology and Immunohistochemistry ducers: erastin, RSL3, FIN56, and FINO215,39,40 (Figure 1D). Formalin-fixed paraffin-embedded (FFPE) tissue sections In contrast, the drugs had no protective effects on apoptosis were stained with hematoxylin/eosin and Masson’stri- inducers: 17-AAG, staurosporine, menadione, UV irradiation, chrome.36 For immunohistochemistry, FFPE sections were and methylglyoxal (Figure 1E, Supplemental Figure 1). Results immunolabeled using anti-hexanoyl-lysine adduct (HEL) of the dose-response relationship and the effective concentra- (1:50, JaICA, 5F12), anti–4-hydroxynonenal (4-HNE) (1:10, tion range of each drug for preventing ferroptosis are shown in JaICA, HNEJ-2), and anti–KIM-1 antibodies (1:400, AF1817; Figure 1F and Supplemental Table 2, respectively. Specifically, R&D). For 4-HNE and KIM-1 staining, sections were heated promethazine, carvedilol, and rifampicin exhibited the high- for antigen retrieval for 5 minutes at 120°C in 0.01 M citrate est activity by preventing BSO-induced ferroptosis at concen- buffer pH 6.0. Terminal deoxynucleotidyl transferase–medi- trations of 0.1, 1, and 3 mM, respectively. In addition, the drugs ated digoxigenin-deoxyuridine nick-end labeling (TUNEL) suppressed the disruption of mitochondrial morphology staining was performed using the ApopTag peroxidase in and the mitochondrial ROS caused by ferroptosis inducers situ apoptosis detection kit (Millipore). The number of (Figure 1G). Our data demonstrated that the positive candi- TUNEL-positive cells was counted per 3200 high-power field. dates among the CYP substrate compounds exerted cell- Visualization of mitochondria in the mouse kidney and liver protective effects specifically in ferroptosis-induced cells. was performed by fluorescence microscopy of an FFPE section stained with Masson’s trichrome.37 The section was observed Antiferroptotic Effects of CYP Substrates Are Caused using a BZ-X800 fluorescence microscope equipped with by Inhibition of Lipid Peroxidation and Not by Their a 3100 objective lens, sectioning module and Texas-Red filter Pharmacologic Activities cube (ex 560/40 nm, em 630/75 nm, dichroic mirror 585 nm). Because ferroptosis is triggered by the accumulation of lipid peroxidation products, we examined whether the positive can- Statistical Analyses didates attenuated this process. BSO treatment stimulated Values are presented as mean6SEM. Statistical analyses were lipid peroxidation as indicated by the increased MDA level conducted using JMP 14 software (SAS Institute). Statistical in the medium and the increased intracellular lipid hydroper- comparisons between groups were analyzed for significance by oxide content (Figure 2, A and B). Treatment with positive two-tailed t test or one-way ANOVA with Dunnett’sorBon- candidates reversed the trend and reduced the levels of MDA ferroni post hoc test. Results were considered significant at and lipid hydroperoxide (Figure 2, A and B). Although P values of ,0.05. the intracellular glutathione content was depleted in both BSO- and erastin-treated cells, the level was not changed by cotreatment with the antiferroptotic compounds (Figure 2C), RESULTS suggesting that these drugs suppressed lipid peroxidation despite glutathione depletion. Screening of CYP Substrates for Antiferroptotic Drugs Next, we investigated the mechanism of lipid peroxida- and Hormones tion inhibition and the corresponding antiferroptotic effect To assess the effect of drugs on ferroptosis, we used glutamine of the drugs. Although the inhibition of LOX activity is com- synthetase inhibitor BSO to induce ferroptosis in H9C2 cells, monly associated with the prevention of lipid peroxidation which are susceptible to ferroptosis.38 Importantly, the sus- and ferroptosis, the positive candidates had either no effect ceptibility of the cells to BSO depended on the assay medium or only a minor LOX inhibitory effect (Figure 2D). The FOX composition used during BSO exposure (Figure 1A). The assay,29 which evaluated the iron-chelating capacity of the higher susceptibility to BSO was observed in the assay medium drugs, indicated that several compounds, especially rifam- containing 1 g/L glucose and 1% FBS, as compared with other picin and omeprazole, can form complexes with ferrous media containing higher glucose and FBS levels. Thus, we used iron (Figure 2E). However, the drugs retained their antifer- the medium containing 1 g glucose/L and 1% FBS during the roptotic activity even in the presence of high ferrous screening. Using the BSO-induced cell death system, we iron concentrations in the medium, unlike the iron chelator screened a total of 30 drugs and hormones that are CYP- deferoxamine that lost the antiferroptotic activity in this inducing and/or CYP substrate compounds (Figure 1B, Sup- medium (Figure 2F), suggesting that the antiferroptotic ac- plemental Table 1). We identified eight drugs and hormones tivity of the CYP substrate drugs did not depend on their that showed antiferroptotic activity, including omeprazole, iron-chelating capacity.

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A Glucose 4.5 g/L + 10% FBS B Glucose 1.0 g/L + 10% FBS BSO(-) BSO (500) Glucose 1.0 g/L + 1% FBS CYP induction or substrate drugs and hormones 1.5 Glu 4.5 g/L 10% FBS Screening 1.0 H9C2 cells Glu 1.0 g/L + BSO (100 µM) 10% FBS + compounds (0.1 -100 µM) 0.5 in Glu 1 g/L + 1% FBS medium

Glu 1.0 g/L

Cell viability (fold change) 8 compounds 0.0 1% FBS prevented cell death 0 5 10 20 50 100 200 500 10002000 BSO [µM]

C + BSO 1.5 *** *** BSO (-) BSO Fer-1 1.5

1.0 1.0

0.5 0.5 Omeprazole Indole-3-C Rifampicin Promethazine Cell viability (fold change) 0 LDH release (OD 490 nm) 0 (µM) DMSO DMSO T3 (10) T3 (10)

BSO (-) BSO (-) Carvedilol PropranololEstradiol T3 Fer-1 (0.1) Fer-1 (0.1) Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (10) Rifampicin (10) Rifampicin (10) Indole-3-C (30) Indole-3-C (30) Propranolol (10) Propranolol (10) Omeprazole (30) Omeprazole (30) Promethazine (1) Promethazine (1) + BSO (100) + BSO (100)

D Erastin E 17AAG RSL3 Staurosporine FIN56 Menadione 1.4 *** FINO2 1.2 *** UV 1.2 1.0 1.0 0.8 0.8 0.6 0.6

propranolol, FINO2) n.s.

* 0.4 0.4 ( 0.2 0.2 Cell viability (fold change) Cell viability (fold change) 0 0 (µM) (µM) DMSO DMSO T3 (10) T3 (10) Inducer (-) Inducer (-) Fer-1 (0.1) Fer-1 (0.1) Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (10) Rifampicin (20) Rifampicin (20) Propranolol (20) Propranolol (20) Indole-3-C (100) Indole-3-C (100) Promethazine (1) Promethazine (1) Omeprazole (100) Omeprazole (100)

Ferroptosis inducer (+) Apoptosis inducer (+)

FGErastin 1.4 Erastin (-) DMSO Promethazine Rifampicin Omeprazole 1.2 Indole-3-C 1.0 Rifampicin 0.8 Promethazine

0.6 Carvedilol Mitotracker 0.4 Propranolol BSO 0.2 Estradiol BSO (-) DMSO Promethazine Rifampicin

Cell viability (fold change) T3 0.0 Fer-1 0.01 0.11 10 100 BSO 100 µM+ Compounds [µM] DHR123

Figure 1. Identification of antiferroptotic drugs and hormones by testing CYP substrate compounds. (A) Viability and images of H9C2 cells treated with BSO for 60 hours using three different medium variations: glucose 4.5 g/L+FBS 10%, glucose 1 g/L+10% FBS, and glucose 1 g/L+1% FBS. n=3. Scale, 100 mm. (B) Flowchart of the screening for antiferroptotic drugs by testing CYP substrate com- pounds. (C) Viability, LDH release, and images of H9C2 cells treated with BSO (100 mM, 60 hours). The compounds were added si- multaneously with BSO. n=6. Scale, 100 mm. (D) Viability of H9C2 cells treated with ferroptosis inducers: erastin (1.5 mM, 24 hours),

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 5 BASIC RESEARCH www.jasn.org

The reported pharmacologic activities of the identified anti- evaluate the scavenging activities of the drugs. The system ferroptotic compounds are diverse. Carvedilol and propranolol demonstrated the dose-dependent scavenging activities are b-adrenergic antagonists, but the knockdown of Adrb1, toward lipid-derived radicals for all drugs except for omepra- which encodes the b1 adrenergic receptor, did not affect their zole, which showed no scavenging activity in this system antiferroptotic activity (Figure 2G). Among the thyroid hor- (Figure 3E). In addition, the antiferroptotic effects of the drugs mones, reverse-T3, which is a physiologically inactive form of on the BSO-induced cell death were strongly associated with T3, also prevented ferroptosis like the other thyroid hormones the scavenging capacity for lipid-derived radicals (R2=0.67), (Figure 2H), suggesting that the ferroptosis-preventing effect compared with that for AAPH radicals (R2=0.14) (Figure 3F). was not mediated by their known pharmacologic activities. Moreover, the NBD-Pen with AA/LOX system showed that the Because we identified the positive candidates among CYP sub- known ferroptosis suppressors Fer-1, liproxstatin-1, and strates, we examined the effect of CYP on the antiferroptotic a-tocopherol also have potent lipid peroxyl radical–scavenging activity. The induction of Cyp1 subfamily is mediated by xe- activities (Figure 3G). Because omeprazole did not have any nobiotic-sensing nuclear receptor Ahr. In Ahr-deficient H9C2 scavenging activity in this experiment, we assumed that the cells generated by CRISPR/Cas9, the induction of Cyp1a1 ex- antiferroptotic effect of omeprazole depended on omepra- pression by the drugs was distinctly reduced (Supplemental zole-derived metabolites that are generated within the cells. Figure 2), but the drugs still showed antiferroptotic activity To test this possibility, we prepared deproteinized cell extracts (Figure 2I). In addition, the antiferroptotic effects of the drugs collected from the cells treated with omeprazole (Figure 3H). were not significantly changed by the treatment with pan- The extracts derived from omeprazole-treated cells showed a CYP inhibitors such as SKF-525A or 1-aminobenzotriazole scavenging activity for lipid peroxyl radicals in the NBD-Pen (Figure 2J). Thus, we concluded that the antiferroptotic ef- assay with the AA/LOX system (Figure 3I). Similarly, the ex- fects of these drugs and hormones were independent of their tracts derived from the cells incubated with the other drugs CYP-associated activities. exhibited significant scavenging activities for lipid peroxyl rad- icals (Figure 3, I and J). These findings demonstrated Antiferroptotic Compounds Function as Lipid Peroxyl that the drugs or their metabolites functioned as lipid peroxyl Radical Scavengers radical scavengers within the cells, which contributed to the To investigate the potential antiferroptotic mechanism of the prevention of ferroptosis by suppressing lipid peroxidation. drugs, we next evaluated the radical-scavenging potentials. Scavenging activities toward superoxide and hydroxyl radicals, Increase of Lipid Peroxyl Radicals Diminishes the which are both major cytotoxic radicals, were examined using Antiferroptotic Activity of the Drugs the ESR–spin trap method with DMPO as a trapping agent. To further study the antiferroptotic mechanism of the drugs, The drugs showed no or only slight scavenging activity toward we examined the effect of an overloaded lipid peroxyl radical superoxide and hydroxyl radicals (even at a concentration of pool on the induction of ferroptosis and the protective effects 100 mM), as compared with that of ascorbate (Figure 3A, Sup- of the drugs. To increase the lipid peroxyl radical pool, we plemental Figure 3). In contrast, the drugs showed a high supplemented the assay with PCOOH which is an oxidation scavenging activity toward peroxyl radicals generated by product of phosphatidylcholine, which is a major component AAPH, whereas the compounds lacking antiferroptotic activ- of mammalian membrane lipids. We specifically prepared ity had a lower scavenging activity toward AAPH-peroxyl rad- 1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3- icals (Figure 3B). However, AAPH-derived peroxyl radicals are phosphocholine which is a dominant form in the human artificial radicals that are not generated under physiologic con- plasma PCOOH pool.41 The results using the in vitro NBD- ditions; therefore, we further investigated the scavenging Pen system demonstrated that PCOOH generated phosphati- activities of the drugs using lipid peroxyl radicals, which are dylcholine peroxyl radical (PCOOc) in the presence of ferrous key molecules in the chain reaction during the progression of iron (Figure 4A). We previously showed that PCOOH added lipid peroxidation. as a supplement to the culture medium was taken up by the To detect lipid peroxyl radicals, we used NBD-Pen20 as a cells.33 Indeed, using the NBD-Pen staining, we observed fluorescence probe for lipid-derived radicals. NBD-Pen de- that the PCOOH treatment increased cellular lipid radicals tected the production of lipid peroxyl radicals using AAs (Figure 4B). Consequently, the PCOOH treatment dose- and the LOX system (Figure 3, C and D), which was used to dependently enhanced the susceptibility to induction of

RSL3 (30 nM, 24 hours), FIN56 (300 nM, 48 hours), and FINO2 (5 mM, 24 hours). n=3. Indicated compounds were added 1 hour before the treatment of inducers. (E) Viability of H9C2 cells treated with apoptosis inducers: 17-AAG (0.3 mM), staurosporine (10 nM), menadione (50 mM), and UV (200 mJ/cm2). n=6 (F) Dose-dependent antiferroptotic effect of the compounds evaluated in BSO-treated H9C2 cells. n=3. (G) Mitochondrial morphology and mitochondrial ROS in H9C2 cells. Erastin (1.5 mM, 20 hours), BSO (100 mM, 24 hours), prom- ethazine (1 mM), and rifampicin (20 mM) were used. Scale, 5 mm. P values were determined by one-way ANOVA, Dunnett’s. *P,0.05, ***P,0.001. Data are mean6SEM. Glu, glucose; UV, ultraviolet.

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A B + BSO (100) 120 *** BSO (-) BSO Omeprazole Indole-3-C Rifampicin 100 80 60

40 Liperfluo 20 MDA in medium (nM) 0 Promethazine Carvedilol Propranolol Estradiol T3 (µM) DMSO T3 (10) BSO (-) Carvedilol (1) Estradiol (10) Rifampicin (20) Indole-3-C (30) Propranolol (10) Omeprazole (30) Promethazine (1) + BSO (100) C D *** 2.5 *** 2.5 *** 100

2.0 2.0 80

1.5 1.5 60

1.0 1.0 40

0.5 0.5 20 GSH (nmol/well)

0 0 15-LOX inhibitory rate (%) 0 1 3 1 3 10 30 10 30 10 30 30 10 30 30 10 10 30 100 (µM) (µM) (µM) T3 DMSO DMSO T3 (10) T3 (10) BSO (-) Erastin(-) NDGA DMSO Estradiol Carvedilol Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (10) Rifampicin Indole-3-C Propranolol Rifampicin (20) Rifampicin (20) Omeprazole Propranolol (10) Propranolol (10) Indole-3-C (100) Indole-3-C (100) Promethazine Omeprazole (30) Omeprazole (30) Promethazine (1) Promethazine (1)

+ BSO (100) + Erastin (1)

EFBSO (-) G BSO BSO + Fe(NH ) (SO ) 0.6 *** 4 2 4 2 siCont siAdrb1 2.0 # 1.4 *** *** 1.2 # *** #

1.5 *** 0.4 *** 1.0 *** ***

# 0.8 1.0

*** 0.6 -XO complex) 0.2 3+ OD 560 nm # 0.5 0.4

(=Fe 0.2

0.0 Cell viability (fold change) 0.0 Cell viability (fold change) 0.0 (-) T3 3 (µM) (µM) DFO DMSO DMSO DMSO DMSO T3 (10) BSO (-) BSO (-) FeCl Estradiol DFO (30) Carvedilol Rifampicin Indole-3-C Propranolol Omeprazole Carvedilol (1) Carvedilol (1) Carvedilol (1) Promethazine Rifampicin (20) Propranolol (10) Propranolol (10) Omeprazole (30) Promethazine (1) + FeCl3 + BSO + BSO

HIAhr KO H9C2 J *** 1.5 (-) 1.5 *** 1.2 *** SKF-525A 1.0 1-ABT 1.0 1.0 0.8 0.6 0.5 0.4 0.5 0.2 Cell viability (fold change)

0.0 Cell viability (fold change) 0.0 0.0 Cell viability (fold change) (µM) (µM) (µM) DMSO DMSO DMSO T3 (10) T3 (10) BSO (-) BSO (-) L-T3 (30) D-T3 (30) Erastin (-) Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (10) Thyroxine (30) Rifampicin (20) Rifampicin (20) Indole-3-C (30) Indole-3-C (30) Propranolol (10) Propranolol (10) Reverse-T3 (30) Omeprazole (30) Omeprazole (30) Promethazine (1) Promethazine (1) + Erastin (1.5) + BSO (100) + BSO (100)

Figure 2. Inhibition of cellular lipid peroxidation by antiferroptotic drugs is not mediated by CYP-related activities. (A) MDA levels in medium after BSO treatment of H9C2 cells (100 mM, 60 hours). n=3. (B) Imaging of intracellular lipid hydroperoxides detected by Liperfluo in BSO-treated H9C2 cells (100 mM, 40 hours). Scale, 50 mm. (C) Intracellular GSH levels after treatment with BSO (100 mM, 24 hours) and erastin (1 mM, 12 hours) for H9C2 cells. n=3. (D) Inhibiting activity to 15-LOX. Nordihydroguaiaretic acid (NDGA) is a

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 7 BASIC RESEARCH www.jasn.org ferroptosis by BSO (Figure 4C). Distinctly, in the presence of acute tubular damage, and evidence of disrupted mitochon- 20 mM PCOOH, the drugs had a diminished antiferroptotic drial morphology in the proximal tubular cells (Figure 6, effect in the BSO-induced cell death (Figure 4D). However, B–D). The oral treatment with promethazine and rifampicin high drug doses were able to prevent the ferroptosis even dur- for 4 days significantly mitigated the renal function decline ing PCOOH treatment because of the reducing effect of their and diminished the renal tubular damage, including the mor- scavenging activities on cellular lipid radicals (Figure 4, E–G). phologic changes of mitochondria. The treatment also re- These findings suggest that excessive lipid peroxyl radical pro- duced the renal lipid peroxidation demonstrated by the duction is involved in ferroptosis induction and that the scav- amounts of detectable 4-HNE and HEL, which are lipid per- enging activity of the drugs for lipid peroxyl radical is responsible oxidation products (Figure 6C), and reduced the number of for the antiferroptotic effects. tubular cell deaths indicated by TUNEL staining (Figure 6E). In addition, the promethazine treatment significantly in- The Antiferroptotic Effects of the Drugs on Cells creased the survival rate in the AKI mice (Supplemental Derived from Various Organs and Species Figure 5). Next, to determine the timing of drug intervention Because cellular lipid peroxidation and susceptibility to fer- that is most effective to protect against AKI, we administered roptosis have been reported to vary among cell lines with dif- promethazine to the cisplatin-injected mice at different time ferent properties,42 we tested the antiferroptotic activity of points (Figure 6F). Intriguingly, all of the groups that received the drugs to BSO- or erastin-induced death in a number of pretreatment with promethazine showed significant amelio- cell lines: NRK49F, HK-2, HUPEC, C2C12, Panc-1, and ration of renal dysfunction and renal tubular damage (Figure 6, MDA-MB-231. We found that the drugs prevented ferroptosis G and H). In contrast, promethazine treatment that began in a variety of cells including the kidney component cells and 24 hours after cisplatin injection did not ameliorate the damage, cancer cells regardless of the tissue type or species (Figure 5A), suggesting that the prevention of lipid peroxidation by although the drugs did not rescue the inhibition of cell pro- a prophylactic administration of the antiferroptotic drug is nec- liferation caused by the high concentration of erastin that was essary to prevent AKI progression. required to induce ferroptosis in the cancer cell lines (Supple- To support that ferroptosis plays a role in cisplatin-induced mental Figure 4). In addition, the antiferroptotic effects were nephrotoxicity, we examined the effect of Fer-1 in the cis- also detected in HT22 mouse neuronal cells treated with high platin-induced AKI mice (Supplemental Figure 6). In the extracellular concentrations of glutamate to induce ferroptosis1 Fer-1–treated group, the levels of creatinine and BUN tended (Figure 5B). to be lower compared with the vehicle group (P=0.06 and 0.07, To further explore the drug candidates, we tested the respectively), suggesting a potential renoprotective effect of antiferroptotic activity of phenothiazine drugs and rifamycin Fer-1 in the model. To examine the effects of the antiferrop- derivatives to which promethazine and rifampicin belong, re- totic drugs on cisplatin-induced cell damage in vitro, we used spectively (Figure 5D). In BSO-treated H9C2 cells, all pheno- the kidney proximal tubular cell lines LLC-PK1 and NRK52E thiazine drugs inhibited ferroptosis, especially promethazine (Supplemental Figure 7). Promethazine and Fer-1 did not and phenothiazine, which had the strongest protective effects. show protective effects on cell damage treated with cisplatin Furthermore, all rifamycin derivatives possessed antiferrop- alone (Supplemental Figure 7, A and B). However, RSL3, totic activity, and the highest activity was associated with which is an inducer of ferroptosis through GPX4 inhibitory rifampicin and rifampicin-quinone. action, synergistically enhanced the cell damage by cisplatin (Supplemental Figure 7A). In GPX4-suppressed conditions Lipid Peroxyl Radical–Scavenging Compounds caused by RSL3, promethazine and Fer-1 showed protective ef- Ameliorated AKI in Mice fects on the cells treated with cisplatin (Supplemental Figure 7, To evaluate the therapeutic potency of the antiferroptotic A-C). In an in vivo setting, the expression of GPX4 was lower drugs in vivo, we tested their efficacy in cisplatin-induced in the kidneys of the cisplatin-injected mice (Supplemental AKI model mice (Figure 6A). Cisplatin injection caused severe Figure 7D), as previously reported.43 These findings suggest renal dysfunction and tubular damage indicated by elevated the possibility that, in an in vivo setting, cisplatin causes an plasma creatinine and BUN levels, along with histopathologic accumulation of lipid peroxidation, possibly via decreased changes including increased KIM-1–positive area, indicating GPX4 expression, and resultant ferroptosis in the kidney, and

pan-LOX inhibitor. n=4. (E) Ferrous ion–chelating effects of the compounds were evaluated by FOX assay in FeCl3 (100 mM) and the compounds (100 mM). The OD 560 nm, which monitors the formation of the Fe3+/xylenol orange complex, was measured at 10 minutes. Deferoxamine (DFO) is an iron chelator. n=3. (F) Viability of H9C2 cells treated with BSO (200 mM, 60 hours) in iron-loaded medium containing 100 mMFe(NH4)2(SO4). n=3. (G) The antiferroptotic effect of carvedilol and propranolol in Adrb1 knockdown H9C2 cells treated with 100 mMBSO.n=3. (H) The antiferroptotic effect of thyroid hormones in H9C2 cells treated with erastin (1.5 mM, 24 hours). n=3. (I) The antiferroptotic effect of the drugs in Ahr KO H9C2 cells treated with 100 mMBSO.n=3. (J) The antiferroptotic effect of drugs in H9C2 cells treated with 100 mM BSO and CYP inhibitors: SKF-525A (10 mM) or 1-aminobenzotriazole (1-ABT, 1 mM). n=3. P values were de- termined by one-way ANOVA, Dunnett’s. ***P,0.001 and ###P,0.001 versus DMSO. Data are mean6SEM.

8 JASN JASN 31: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

DMPO-OOH for O2- A DMPO-OH for OH• B 120 14 100 12 1 µM 3 µM 80 10 10 µM 8 60 100 µM 6 40 4 20

AAPH peroxyl radical 2 ESR signal intensity intensity (%) ESR signal 0 0 absorbance capacity (/µMabsorbance capacity TE) T3 T3 Blank DHEA Estradiol Estradiol Carvedilol Phenytoin Carvedilol Ascorbate Indole-3-C Rifampicin Indole-3-C Rifampicin Propranolol Propranolol Aldosterone Amitriptyline Omeprazole Omeprazole Testosterone Pentobarbital Promethazine Promethazine Carbamazepine Dexamethasone

Each 100 µM Anti-ferroptotic drugs Compounds without antiferroptotic effect C E LOX Promethazine Rifampicin Indole-3-C Carvedilol LH LOOH 0(µM) 0 30 30 (µM) 30 30 (AA) 30 1 0 100 0 3 3 3 20 10 20 20 300 20 LOO • 1000 10 30 10 30 10 10 10 (µM) 10 NBD-Pen 100 LOX-dependent 30 Fluorescence (a.u.) FL intensity 0 100 0 100 0 0 (µM) 012345 012345 012345 012345

D Propranolol Estradiol T3 Omeprazole 30 100 50 AA + LOX 0 30 30 50 30 300 1000 40 30 0 0 50 3 0 20 20 3 30 20 100 10 (µM) 25 20 30 10 (µM) AA 10 10 (µM) 10 30 10 LOX

FL intensity (a.u.) FL intensity (µM) 0 Blank 0 0 0 0 012345 0 12345 012345 012345 012345 Time (m) Time (m) Time (m) Time (m) Time (m) F G 2 2 Ferrostatin-1 Liproxstatin-1 -Tocopherol R = 0.67 R = 0.14 100 15 0 0 0 Indole-3-C Propranolol Estradiol 1 40 10 Carvedilol 30 1 30 30

15% of 10 Rifampicin 3 30 (µM)

 100 10 T3 20 (µM) 20 Estradiol 20 3 (µM) Promethazine 5 Promethazine Carvedilol Indole-3-C 10 10 10 intensity (a.u.) intensity NBD-FL (µM) T3 AAPH radical Propranolol 10 Rifampicin 10 LOX-dependent FL Conc. for 1 0 0 30 0 30 0 0510absorbance capacity 0510 0 12345012345 012345 of 10 µM drugs (/µM TE) EC50 in BSO cell death (µM) EC50 in BSO cell death (µM) Time (m) Time (m) Time (m)

H I Extracts of cells Extracts of cells J DMSO treated with treated with ** or DMSO DMSO Drugs 200 200 200 Indole-3-C (100) Omeprazole Rifampicin (30) 100 Promethazine (3) m at 5 Incubation 100 100 Deproteinized Cell extracts (-) Carvedilol (3) FL intensity FL intensity (a.u.) 0 cell extracts LOX-dependent

FL intensity FL intensity (a.u.) Propranolol (30) + 0 0

Estradiol (20) (µM) AA/LOX 012345 012345 DMSO Time (m) Time (m) T3 (20) T3 (20) NBD-Pen (µM) Carvedilol (3) Carvedilol Estradiol (20) Estradiol

Fluorescence (30) Rifampicin Propranolol (30) Propranolol Indole-3-C (100) Promethazine (3) Omeprazole (100)

Figure 3. The antiferroptotic drugs scavenge lipid peroxyl radicals. (A) Analysis of scavenging potential to superoxide and hydroxyl radicals evaluated by ESR–spin trapping. ESR signal intensities of DMPO-OOH for superoxide determination and DMPO-OH for hy- droxyl radical determination. Effects of the compounds (each 100 mM) are shown. Each value represents the mean of duplicate measurements. (B) The absorbance capacity to AAPH-derived peroxyl radicals. Effects of compounds (1, 3, 10, and 100 mM) are shown as Trolox equivalent (TE). (C) Experimental scheme for detection of lipid peroxyl radicals using NBD-Pen and AA/LOX system. AA, arachidonic acids; LH, lipid molecule; LOOH, lipid hydroperoxide; LOOc, lipid peroxyl radical. (D) Fluorescence response of NBD-Pen to lipid peroxyl radicals generated by AA/LOX system. FL, fluorescence intensity at ex/em=470/530 nm. (E) Scavenging activities to lipid peroxyl radicals evaluated by fluorescence intensities of NBD-Pen. Representative data of three experiments were shown.

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 9 BASIC RESEARCH www.jasn.org that this was ameliorated by the treatment with antiferroptotic radical-trapping agents. Therefore, our results (1)indicate drugs. that evaluation of the lipid peroxyl radical–scavenging effect We also tested the therapeutic potency of the antiferrop- offers an efficient approach to assess the activity of antiferrop- totic drug using LPS/GalN-induced acute liver injury model totic drugs, and (2) reveal the substantial mechanism by which mice (Figure 7A). LPS/GalN induced severe liver damage, radical-trapping compounds exert antiferroptotic effects. diffuse hepatocyte cell death, hemorrhaging, and swelling of Because the identified compounds are US Food and Drug Ad- mitochondria (Figure 7, B–E). Oral promethazine treatment ministration–approved drugs, they can be subjected to targe- significantly protected against the LPS/GalN-induced patho- ted testing for new therapeutic applications in a wide range of logic changes, reduced the elevated plasma ALT level and the ferroptosis-related diseases, including renal pathologic number of TUNEL-positive cell deaths, and prevented tissue conditions. lipid peroxidation according to the results of HEL staining. Although we identified the antiferroptotic drugs by screen- Our findings indicated that the drugs prevented the progres- ing CYP-inducing compounds and/or CYP substrates, the sion of lipid peroxidation in the affected organs and inhibited effects for preventing ferroptosis did not depend on their subsequent ferroptosis, resulting in the protection from acute CYP-related effects or reported pharmacologic activities. Al- organ failure. though numerous compounds are CYP substrates, they are typically lipophilic organic compounds with relatively low molecular weight.44 These preferred properties of CYP sub- DISCUSSION strates, such as lipophilicity, ease of oxidation, and chemical reactivity, are characteristics that would also be associated We identified a variety of ferroptosis-preventing drugs and with the ability to function as lipid radical scavengers. Most hormones by the screening of CYP substrate compounds. of the identified drugs were previously reported to show lipid The mechanism involved the inhibition of lipid peroxida- peroxidation–preventing effects that were considered to be tion by scavenging the lipid peroxyl radicals that promote initiated via their antioxidant functions.45–50 Our results in- the induction of ferroptosis. The drugs also exerted the anti- dicate that lipid peroxyl radical–scavenging activities are re- ferroptotic effects in the kidney component cells and sup- sponsible for these effects, although iron-chelating activity pressed acute organ injuries (Figure 7F). Thus, our findings could also contribute in carvedilol.29 Furthermore, our find- indicate the importance of lipid peroxyl radical accumula- ings elucidated the mechanism of cytoprotective effects that tion in ferroptosis induction, and that the pharmacologic has been incompletely described for some CYP substrate drugs reduction of lipid peroxyl radicals, such as by the repurposed and hormones, in earlier reports.51,52 Therefore, when a com- drugs, prevents ferroptosis-related disorders, including pound possesses cell-protective activities specific for ferrop- AKI. tosis, we need to consider the possibility that the mechanism is In this study, we showed that the identified antiferroptotic mediated by its lipid radical–scavenging ability independent of drugs possess scavenging activities for lipid peroxyl radicals. It its original pharmacologic activity. Despite both ferroptosis has been known that radical-trapping antioxidants can pre- and apoptosis being considered ROS-dependent cell death, vent ferroptosis. However, we showed that the antiferroptotic drugs with antiferroptotic activity did not prevent apoptotic effects of the drugs were closely associated with the scavenging cell death. This may be attributed to their different scavenging of lipid peroxyl radicals and not much related to interactions affinities to the radical species. The drugs showed high scav- with other radicals such as hydroxyl radical, superoxide, or enging activity against lipid radicals, which induce ferroptosis, artificial radicals. In addition, our lipid radical detection but did not show high scavenging ability against hydrophilic method using NBD-Pen provided direct evidence that known radicals, such as hydroxy radicals, which are known to damage ferroptosis inhibitors Fer-1 and liproxstatin-1 also exerted mitochondria and cause apoptosis. Although we performed a strong scavenging activities for lipid peroxyl radicals. Further- relatively small-scale screening experiment to identify com- more, our findings suggest that the increased level of cellular pounds with antiferroptotic effect, up to one-quarter of the lipid peroxyl radicals appears to be a critical parameter for tested compounds were positive hit candidates and a total of the onset of ferroptosis and that the reduction of this radical 20 compounds were identified as antiferroptotic drugs in our level by scavenging is the antiferroptotic mechanism of the study. Thus, a large-scale effort targeting CYP substrates and

(F) Drugs were evaluated for correlation between antiferroptotic effect (evaluated by EC50 in BSO-induced cell death) and lipid peroxyl radical–scavenging activity (evaluated by 15% reduction of NBD-Pen FL intensity in AA/LOX system), and AAPH-radical–scavenging activity (evaluated by the effect of the 10-mM drugs). (G) The scavenging activities of known ferroptosis inhibitors of lipophilic antioxidants evaluated by NBD-Pen using the AA/LOX system. (H) Experimental schema for evaluation of scavenging activity to lipid peroxyl radicals using deproteinized extracts from the drug-treated H9C2 cells. (I and J) Fluorescence response of NBD-Pen with the extracts of cells incubated with DMSO, omeprazole (100 mM), or other drugs for 12 hours. The values at 5 minutes were shown in (J). n=3. P values were determined by one-way ANOVA, Dunnett’s. **P,0.01. Data are mean6SEM. DMPO-OH, 2-Hydroxy-5,5-dimethyl-1-pyrrolidinyloxy; DMPO-OOH, 2,2-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl.

10 JASN JASN 31: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

AB PCOOH (50 µM) + Fe2+ 100 PCOOH PCOOH PCOOH 80 Fe2+ PCOOH (-) (20 µM) (50 µM)

60 PCOO • PCOOH (15) + Fe2+ NBD-Pen NBD-pen 40

FL intensity (a.u.) 2+ Fluorescence 20 PCOOH (0) + Fe PCOOH (50) NBD-Pen Hoechst 0 PCOOH (15) no Fe2+ 03691215 PCOOH (0) Time (m) C E BSO + RIF BSO + PMZ 1.2 PCOOH 0 µM BSO + RIF + PCOOH BSO + PMZ + PCOOH 1.0

PCOOH 10 µM ** 1.2 1.2

0.8 PCOOH 20 µM *** 1.0 1.0 0.6 0.8 0.8 PCOOH 50 µM 0.6 0.6 0.4 * ** 0.4 0.4 0.2 *** 0.2 0.2 * * * Cell viability (fold change) 0.0 0.0 0.0

0 1 3 10 30 100 Cell viability (fold change) 0 3 10 30 0 0.1 0.3 1 3 10 BSO [µM] RIF [µM] PMZ [µM]

D F BSO PCOOH (-) PCOOH (20 µM) BSO (-)BSO + RIF (10) + PMZ (0.3) + PMZ (3) (µM) 1.5 * * * * * * * * 1.0

0.5

0.0 + PCOOH PCOOH (-) Cell viability (fold change) (µM)

T3 (5) G

DMSO PCOOH BSO (-) PCOOH (-) DMSO PMZ (3 µM) RIF (30 µM) Estradiol (5) Rifampicin (10) Indole-3-C (30) Carvedilol (0.3) Propranolol (10) Omeprazole (30) Promethazine (0.3) BSO (100) NBD-Pen

Figure 4. Lipid peroxyl radical promotes ferroptosis and diminishes the antiferroptotic effects of the drugs. (A) Fluorescence response of NBD-Pen to PCOOH (0, 15, 50 mM) with or without Fe2+. PCOOH in the presence of Fe2+ generated PCOOc detected by NBD-Pen. FL, fluorescence intensity. (B) Cellular lipid radicals imaging detected by NBD-Pen in BSO (100 mM, 12 hours)-treated H9C2 cells with or without PCOOH. Scale, 50 mm. (C) Cell viability of BSO (indicated dose, 48 hours)-treated H9C2 cells incubated with PCOOH. n=3. (D) The antiferroptotic effects of the drugs in BSO (100 mM, 60 hours)-treated H9C2 cells incubated with or without PCOOH (20 mM). n=3. (E and F) Cell viability and images of BSO (100 mM, 60 hours)-treated H9C2 cells incubated with promethazine (PMZ) or rifampicin (RIF) with or without PCOOH (20 mM). n=3. Scale, 100 mm. (G) Cellular NBD-Pen signals in the presence of PMZ or RIF in BSO (100 mM, 12 hours)-treated H9C2 cells with PCOOH (20 mM, 1 hour). Scale, 50 mm. P values were determined by one-way ANOVA, Dunnett’s (in C), and Bonferroni (in D and E). *P,0.05, **P,0.01, ***P,0.001. Data are mean6SEM. lipid peroxyl radical–scavenging ability is predicted to identify relationship between each CYP subfamily substrate and their many more antiferroptotic drugs. However, the CYPs include scavenging ability. .40 subfamilies that act on a large and diverse array of sub- We showed that antiferroptotic drugs ameliorated cis- strates; thus, future research should assess the structure-activity platin-induced AKI and LPS/GalN-induced liver injury.

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 11 BASIC RESEARCH www.jasn.org

A NRK49F HK-2 HUPEC 1.2 *** 1.2 1.2 ** *** 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 Cell viability (fold change) (µM) T3 (3) DMSO DMSO DMSO T3 (10) T3 (10) BSO (-) Erastin (-) Erastin (-) Estradiol (20) Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (20) Rifampicin (20) Rifampicin (20) Rifampicin (10) Indole-3-C (50) Indole-3-C (30) Propranolol (10) Propranolol (10) Indole-3-C (100) Omeprazole (30) Omeprazole (30) Omeprazole (30) Promethazine (1) Promethazine (1) Promethazine (1) BSO (100) Erastin (5) Erastin (0.5)

C2C12 Panc-1 MDA-MB-231 1.2 *** 1.2 1.2 1.0 1.0 ** 1.0 ** 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 Cell viability (fold change) (µM) DMSO DMSO DMSO T3 (10) T3 (10) T3 (10) BSO (-) Erastin (-) Erastin (-) Carvedilol (1) Carvedilol (1) Carvedilol (1) Estradiol (20) Estradiol (10) Estradiol (10) Rifampicin (20) Rifampicin (10) Rifampicin (30) Indole-3-C (30) Indole-3-C (10) Indole-3-C (30) Propranolol (10) Omeprazole (30) Omeprazole (30) Promethazine (1) Promethazine (1) Promethazine (1) BSO (200) Erastin (5) Erastin (10) B HT22 HT22 1.2 *** 0.5 *** 1.0 0.4 0.8 0.3 0.6 0.2 0.4 0.2 0.1 0.0 0 Cell viability (fold change) LDH release (OD 490 nm) (µM) (µM) DMSO DMSO T3 (10) T3 (10) Glutamate (-) Glutamate (-) Carvedilol (1) Carvedilol (1) Estradiol (10) Estradiol (10) Rifampicin (30) Rifampicin (30) Indole-3-C (30) Indole-3-C (30) Propranolol (10) Propranolol (10) Omeprazole (30) Omeprazole (30) Promethazine (0.3) Promethazine (0.3) Glutamate (4 mM) Glutamate (4 mM) C Phenothiazine drugs Rifamycin drugs 1.4 1.2 Promethazine Rifampicin 1.2 Phenothiazine 1.0 Rifampicin-quinone 1.0 Perphenazine 0.8 0.8 Rifabutin Promazine 0.6 0.6 Rifapentin 0.4 Prochlorperazine 0.4 Rifaximine 0.2 Chlorpromazine 0.2 0.0 0.0 Cell viability (fold change) 0.01 0.03 0.1 0.3 1 3 10 0.3 1 3 10 30 100 BSO 100 µM + Compounds [µM] BSO 100 µM + Compounds [µM]

Figure 5. The antiferroptotic effects of the drugs on cells derived from various organs and species. (A) Antiferroptotic effect of the drugs on various cell lines: NRK49F (100 mM BSO, 96 hours), HK-2 (5 mM erastin, 48 hours), HUPEC (0.5 mMerastin,48hours),C2C12(200mM BSO, 60 hours). Panc-1 (5 mM erastin, 30 hours), and MDA-MB-231 (10 mM erastin, 48 hours). n=3. (B) Prevention of glutamate-induced neuronal cytotoxicity in HT22 cells by drugs. Cells were exposed to 4 mM glutamate for 24 hours. n=3. (C) Antiferroptotic effects of phenothiazine-derived drugs and rifamycin derivatives evaluated in BSO (100 mM)-treated H9C2 cells. n=3. P values were determined by one-way ANOVA, Dunnett’s. *P,0.05. Data are mean6SEM.

12 JASN JASN 31: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

A (n) 0 h 96 h C Cis (-) Cis + vehicle Cis + PMZ Cis + RIF (3) Cisplatin (8)

+ PMZ (every 12h) Masson trichrome (8) + RIF (every 12h) (8) KIM-1 ** B 1.2 * ** 1.0 0.8 HEL 0.6 0.4 0.2 4-HNE Creatinine (mg/dL) 0.0

PMZ RIF Cis (-) Cis + vehicle Cis + PMZ Cis + RIF Cis (-) vehicle D + Cisplatin ** 250 ***** 200 Mitochondria 150

100 High BUN (mg/dL) 50 magnification 0 *** 12 *** *** PMZ RIF Cis (-) vehicle 8 E + Cisplatin tubules/HPF

Cis (-) Cis + vehicle Cis + PMZ Cis + RIF + 4

TUNEL 0

TUNEL s (-) hicle RIF i e PMZ C v + Cisplatin F G *** ** ** ** (n) 0 12 24 96 h 0.8 *** *** 250 * * Cisplatin (-) (3) 200 Cisplatin 0.6 (7) 150 + PMZ at pre 30 min 0.4 100 (7)

0.2 BUN (mg/dL) + PMZ pre - 24 h 50 Creatinine (mg/dL) (6) 0 0 e + PMZ 24 - 96 h (-) l (-) s (7) Ci Cis vehic Z at pre vehicle M + PMZ pre - 96 h P PMZ at pre (7) PMZ prePMZ – 24 24hPMZ – 96 pre h – 96 h PMZ prePMZ – 24 24 hPMZ – 96 pre h – 96 h + Cisplatin + Cisplatin

H + Cisplatin Cis (-) vehicle PMZ at pre PMZ pre-24 h PMZ 24-96 h PMZ pre-96 h Masson trichrome

Figure 6. The antiferroptotic drugs ameliorated cisplatin-induced AKI. (A) Experimental scheme of cisplatin-induced AKI. Promethazine (PMZ; 20 mg/kg) and rifampicin (RIF; 20 mg/kg) were orally administered every 12 hours. (B) The levels of BUN and plasma creatinine. (C) Histologic images of kidneys of Masson trichrome staining and immunohistochemistry for KIM-1, HEL, and 4-HNE. Scale, 100 mm. (D) Mitochondria imaging in the proximal tubular cells. Scale, 5 mm. (E) TUNEL staining. Scale, 100 mm. (F) Experimental scheme 2. PMZ (20 mg/kg) was ad- ministered at the indicated points in each group. (G and H) The levels of BUN and creatinine, and histologic images of kidneys. Scale, 100 mm. P values were determined by one-way ANOVA, Dunnett’s. *P,0.05, **P,0.01, ***P,0.001. Data are mean6SEM. Cis, cisplatin.

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A B C LPS/GalN (-) LPS/GalN LPS/GalN + PMZ

2000 ** * HE

(n) h h-12 h-2 6 h0 1500 (4) LPS/GalN 1000 (8)

+ PMZ ALT (IU/L) 500 HE high

(10) magnification 0 HEL +PMZ LPS/GaINLPS/GaIN LPS/GaIN (-)

D LPS/GalN (-) LPS/GalN LPS/GalN + PMZ E LPS/GalN (-) LPS/GalN LPS/GalN + PMZ TUNEL Mitochondria

** ** 60 cells High + 40 /HPF 20 magnification TUNEL 0

+PMZ LPS/GaINLPS/GaIN LPS/GaIN (-) F Oxidative stress Depletion of GSH Inhibition of GPX4 LOX LH LOOH Fe2+ Anti-ferroptotic CYP-substrate action radical drugs and hormones LOO • LH L • LOO • Chain Scavenging reaction Prevention from Suppression of ferroptosis lipid peroxidation

LOOH LH Lipid peroxidation Organ protection Ferroptosis Organ failure

Figure 7. Promethazine ameliorated acute liver damage by LPS/GalN. (A) Experimental scheme of LPS/GalN-induced acute liver in- jury. (B) The level of plasma ALT. (C) Histologic images of livers of hematoxylin/eosin (HE) staining and immunohistochemistry for HEL. Scale, 500 mm for HE and 100 mm for HE high magnification and HEL. (D) TUNEL staining. Scale, 100 mm. (E) Mitochondria imaging in the hepatocytes. Scale, 5 mm. (F) The mechanism of the antiferroptosis effect of drugs identified by screening of CYP substrate compounds. P values were determined by one-way ANOVA, Dunnett’s. *P,0.05, **P,0.01. Data are mean6SEM. ALT, alanine aminotransferase; LH, lipid molecule; Lz, lipid radical.

14 JASN JASN 31: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

In previous reports, iron chelators prevented lipid peroxida- pathway other than ferroptosis are involved in the pathophys- tion and ameliorated renal damage in cisplatin nephrotoxi- iology.63 However, there have been no established specific city,53,54 also suggesting that ferroptosis is involved in the markers to demonstrate the presence of ferroptosis in vivo, pathophysiology of cisplatin-induced AKI. Although, in this such as cleaved caspase-3 in apoptosis. Hence, direct evidence study, the renoprotective effect of Fer-1 was not statistically of the attenuation of ferroptosis in vivo is difficult to obtain in significant in the cisplatin model (Supplemental Figure 6), the current situation. The possibility does remain that the recent studies showed that the antiferroptotic effect of Fer-1 suppression of ferroptosis by antiferroptotic drugs might isnotpotentinin vivo conditions, unlike its high activity in also be associated with the prevention of secondary apoptosis vitro, because of its instability in plasma.55 Our results show triggered by ferroptosis under in vivo conditions in which that the renoprotective effect of promethazine was more po- complex cell death communication occurs. A second limita- tent than that of Fer-1, suggesting that promethazine would tion is that we showed the therapeutic efficacy of the drugs be a more useful tool to prevent ferroptosis in in vivo disease only in acute disease models. Even if drugs have antiferrop- models. Indeed, the used dose of 20 mg/kg of promethazine totic effects, if the compounds have chronic cytotoxic effects, and rifampicin is predicted to maintain an effective concen- long-term use may show harmful effects. Indeed, the long- tration for antiferroptotic activity in vivo because oral admin- term use of omeprazole has been associated with an increased istrations of 5 mg/kg promethazine and 20 mg/kg rifampicin risk of CKD in epidemiologic studies.64 were reported to maintain 0.1 and 10 mMoftheirplasma In summary, the lipid peroxyl radical is a key molecule levels, respectively, for approximately 10 hours,56,57 and this during the onset of ferroptosis and the treatment with radical was within the range to prevent ferroptosis in the in vitro anal- scavengers specific for lipid peroxyl radicals, such as certain ysis (Supplemental Table 2). In the LPS-induced endotoxemia CYP substrate drugs, represents a promising pharmacologic model, a previous report showed that Fer-1 treatment did not approach to prevent ferroptosis. Thus, to target lipid peroxyl increase the survival rate7; however, unlike LPS alone, LPS radicals should lead to the development of therapeutics that with GalN prominently enhanced the secretion of inflamma- can control ferroptosis-related kidney diseases. Our results tory cytokines and then caused fulminant hepatic failure with provide insights into the molecular processes associated with 100% lethality within 12 hours in the mice when no protective ferroptosis and suggest a strategy for developing drugs for treatments were performed.58 Thus, the pathophysiology is ferroptosis-associated diseases, including AKI. different between the LPS alone and LPS/GalN models, the latter of which involves massive cell death in the liver. These differences in the models would influence the extent of the therapeutic effects of ferroptosis inhibitors. ACKNOWLEDGMENTS In addition to disease-associated tissue damages, ferropto- sis has been reported to play a potential role in the cytotoxic We acknowledge the support of the Biomedical Research Core of mechanism of some anticancer drugs including cisplatin, sor- Tohoku University Graduate School of Medicine. We are grateful to afenib, and immune checkpoint inhibitors.59–61 Interestingly, Prof. Jeffrey B. Kopp for providing human urine-derived podocyte- sensitivity to ferroptosis of some tumors has been reportedly like epithelial cells, and thank Y. Sasaki for technical assistance and associated with chemosensitivity to certain anticancer drugs.62 Keyence Corporation for the use of a BZX-810 microscope. In our study, the antiferroptotic drugs also prevented cell Dr. Mishima designed the study. Dr. Mishima and Dr. Sato carried death in the cancer cell lines, suggesting that the coadminis- out experiments. Dr. Ito, Dr. Yamada, and Dr. Nakagawa prepared tration of these drugs may weaken the therapeutic effects of materials. Dr. Mishima and Dr. Sato analyzed the data. Dr. Mishima the ferroptosis-mediated anticancer agents. made the figures. Dr. Mishima and Dr. Abe drafted the paper. For cell death experiments in this study, we used an assay Mrs. Suzuki, Dr. Oikawa, Dr. Matsuhashi, Dr. Kikuchi, Dr. Toyohara, medium containing 1.0 g/L glucose and 1% FBS. As well as in Dr. Suzuki, and Dr. Ito contributed to the interpretation of the results. the BSO-induced ferroptosis, the sensitivity to the induction All authors approved the final version of the manuscript. was higher in the medium condition compared with that in the high glucose/10% FBS medium in the erastin- and RSL3- induced ferroptosis (Supplemental Figure 8). FBS contains DISCLOSURES various antioxidant proteins with ferroptotic activities, such as selenoproteins,4 which may exert potential antiferroptotic All authors have nothing to disclose. effects in a medium rich in FBS. Further studies are needed to clarify what factors are responsible for the altered sensitivity to ferroptosis induction under different medium conditions. FUNDING A limitation of this study is that the attenuation of ferrop- fi fi This study was supported by Grants-in-Aid for Scienti c Research from the tosis by the antiferroptotic drugs was not directly con rmed Japan Society for the Promotion of Science (18K08198 and 18H02822) and a in vivo, although the drugs attenuated tissue injury and cell grant from the Japan Foundation for Applied Enzymology, HIROMI Medical death. In the organ injury models, various types of cell death Research Foundation, and AMED CREST (JP19gm0910013).

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 15 BASIC RESEARCH www.jasn.org

SUPPLEMENTAL MATERIAL 11. Guerrero-Hue M, García-Caballero C, Palomino-Antolín A, Rubio- Navarro A, Vázquez-Carballo C, Herencia C, et al.: Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferrop- This article contains the following supplemental material tosis-mediated cell death. FASEB J 33: 8961–8975, 2019 online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ 12. Niki E, Yoshida Y, Saito Y, Noguchi N: Lipid peroxidation: Mechanisms, ASN.2019060570/-/DCSupplemental. inhibition, and biological effects. Biochem Biophys Res Commun 338: Supplemental Table 1. The list of screened compounds. 668–676, 2005 Supplemental Table 2. Antiferroptotic activity and cytotoxicity of 13. Xie Y, Song X, Sun X, Huang J, Zhong M, Lotze MT, et al.: Identification of baicalein as a ferroptosis inhibitor by natural product library the compounds in H9C2 cells. screening. Biochem Biophys Res Commun 473: 775–780, 2016 Supplemental Figure 1. Antiferroptotic drugs did not prevent 14. Zilka O, Shah R, Li B, Friedmann Angeli JP, Griesser M, Conrad M, et al.: methylglyoxal-induced cell death. On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 Supplemental Figure 2. Cyp1a1 induction in WT and AhR KO and the role of lipid peroxidation in ferroptotic cell death. ACS Cent Sci H9C2 cells. 3: 232–243, 2017 15. Shimada K, Skouta R, Kaplan A, Yang WS, Hayano M, Dixon SJ, et al.: Supplemental Figure 3. ESR spectra of DMPO-OOH and Global survey of cell death mechanisms reveals metabolic regulation of DMPO-OH. ferroptosis. Nat Chem Biol 12: 497–503, 2016 Supplemental Figure 4. Antiferroptotic drugs did not rescue in- 16. Yin H, Porter NA: New insights regarding the autoxidation of poly- hibition of cell proliferation by high-dose erastin treatment. unsaturated fatty acids. Antioxid Redox Signal 7: 170–184, 2005 Supplemental Figure 5. Effects of antiferroptotic drugs on survival 17. Feng H, Stockwell BR: Unsolved mysteries: How does lipid perox- idation cause ferroptosis? PLoS Biol 16: e2006203, 2018 rate in cisplatin-induced AKI mice. 18. Skouta R, Dixon SJ, Wang J, Dunn DE, Orman M, Shimada K, et al.: Supplemental Figure 6. Effects of Fer-1 on cisplatin-induced AKI. Ferrostatins inhibit oxidative lipid damage and cell death in diverse Supplemental Figure 7. Protective effect of antiferroptotic drugs disease models. J Am Chem Soc 136: 4551–4556, 2014 on cell damage by cisplatin plus GPX4 inhibition. 19. Chamulitrat W, Mason RP: Lipid peroxyl radical intermediates in the Supplemental Figure 8. Effect of medium condition on ferroptosis peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations. J Biol Chem 264: 20968– induction by erastin and RSL3. 20973, 1989 Supplemental Material. Detailed methods and materials information. 20. 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AFFILIATIONS

Divisions of 1Nephrology, Endocrinology, and Vascular Medicine and 5Pediatrics, and 8Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Department of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan; 3Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; 4Physical Chemistry for Life Science Laboratory, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; 6Katta Public General Hospital, Shiroishi, Japan; and 7Department of Medical Science, Tohoku University Graduate School of Biomedical Engineering, Sendai, Japan

JASN 31: ccc–ccc,2019 Drugs Prevent Ferroptosis and AKI 17 A Table of Supplemental Contents

Supplemental Table 1. The list of screened compounds.

Supplemental Table 2. Anti-ferroptotic activity and cytotoxicity of the compounds in H9C2 cells.

Supplemental Figure 1. Anti-ferroptotic drugs did not prevent methylglyoxal-induced cell death.

Supplemental Figure 2. Cyp1a1 induction in WT and AhR KO H9C2 cells.

Supplemental Figure 3. ESR spectra of DMPO-OOH and DMPO-OH.

Supplemental Figure 4. Anti-ferroptotic drugs did not rescue inhibition of cell proliferation by high-dose

erastin treatment.

Supplemental Figure 5. Effects of anti-ferroptotic drugs on survival rate in cisplatin-induced AKI mice.

Supplemental Figure 6. Effects of Fer-1 on cisplatin-induced AKI.

Supplemental Figure 7. Protective effect of anti-ferroptotic drugs on cell damage by cisplatin plus GPX4

inhibition.

Supplemental Figure 8. Effect of medium condition on ferroptosis induction by erastin and RSL3.

Detailed methods and materials information. Supplemental Table 1

Omeprazole Rifampicin Indole-3-carbinol Carvedilol Propranolol Promethazine Carbamazepine Pentobarbital Phenobarbital Nifedipine Bezafibrate Spironolactone Amitriptyline Imipramine Pioglitazone Rosiglitazone Ritonavir Isoniazid Sulfinpyrazone Phenytoin -Estradiol 3,3',5-Triiodo-L-thyronine (T3) Dexamethasone Prednisolone Aldosterone Testosterone Dihydrotestosterone Dehydroepiandrosterone (DHEA) Dehydroepiandrosterone 3-sulfate Pregnenolone-16α-carbonitrile

Supplemental Table 1. The list of screened compounds Supplemental Table 2

Prevention of Cytotoxicity ferroptosis by BSO in H9C2 in H9C2

Compound EC50 (μM) TC50 (μM) Omeplazole 15.9 136 Indole-3-carbinol 9.1 217 Rifampicin 1.6 195 Promethazine 0.04 8.6 Carvedilol 0.35 6.8 Propranolol 4.6 56 Estradiol 4.8 > 500 T3 4.3 > 500

Supplementary Table2. Anti-ferroptotic activity and cytotoxicity of the compounds in H9C2 cells

Ferroptosis was induced with BSO (100 M, 60 h) and EC50 was calculated by the value of cell viability. Cytotoxicity TC50 was calculated the value of cell viability following 60 h incubation with the compound. n=3 Supplemental Figure 1

A + MG *** 1.2 MG (-) MG (1 mM) Omeprazole Indole-3-C 1.0 0.8 0.6 RifampicinPromethazine Carvedilol Propranolol 0.4 n.s. 0.2

Cell viability (fold Cell viability change) 0 M) 

( Estradiol T3 Fer1 DMSO T3 (10) Fer-1 (0.1) Glutamate (-) Carvedilol (1) Carvedilol Estradiol (10) Rifampicin (10) Indole-3-C (30) Propranolol (10) Omeprazole (30) Promethazine (1) MG (1 mM) B MG (-) MG (1 mM) 1.4 * * * * * * * * 1.2 * 1.0 0.8 0.6 0.4 0.2

Cell viability (fold Cell viability change) 0 M)  ( DMSO T3 (10) Glutamate (-) Carvedilol (1) Carvedilol Estradiol (10) Rifampicin (10) Indole-3-C (30) Propranolol (10) Omeprazole (30) Promethazine (1) BSO (100 μM)

Supplemental Figure 1. Anti-ferroptotic drugs did not prevent methyglyoxal-induced cell death A. Viability and images of H9C2 cells treated with methyglyoxal (MG, 1 mM for 72 h) and indicated compounds. n=3. Scale, 50 μm. B. The protective effect of the drugs in BSO (100 μM, 60 h)-treated H9C2 cells was diminished in the presence of MG. n=3. *P<0.05, ***P<0.001. Data are mean ± S.E.M. Supplemental Figure 2 indicated drugs for 12 h. n=3. * Cyp1a1 c KO H9C2 Figure Supplementary2. induction Cyp1a1 in andAhR WT mRNA expression in wild type H9C2 and AhR KO-H9C2 cells treated cells KO-H9C2 and AhR type in expression H9C2 wild mRNA

Cyp1a1 mRNA/Actb Fold change 100 200 10 20 15 P 5 0 <0.05, **

DMSO P=0.11 Omeprazole(100) **

P Indole-3-C (100) <0.01 (t-test). Rifampicin (100) ** P=0.17 Promethazine (30)

Carvedilol (3) **

Propranolol (100) * ** Estradiol (100)

T3 (100) * (M) h KO Ahr WT ells with Supplemental Figure 3

- A DMPO-OOH for O2 Blank (Acetone) Omeprazole Indole-3-C Rifampicin Promethazine Carvedilol Propranolol Estradiol

Blank (water) Ascorbate

330.5 335.5 340.5 Magnetic field (mT)

B DMPO-OH for OH•

Blank (Acetone) Omeprazole Indole-3-C Rifampicin Promethazine Carvedilol Propranolol Estradiol

Blank (water) Ascorbate

330.5 335.5 340.5 Magnetic field (mT)

Supplementary Figure 3. ESR spectra of DMPO-OOH and DMPO-OH Representative ESR spectra of (A) DMPO-OOH (for superoxide determination) by the hypoxanthine-XOD system and (B) DMPO-OH (for hydroxyl radical determination) generated by the Fenton reaction. Effects of compounds (each 100 M) were shown. Supplemental Figure 4

Erastin (-) Erastin (10 uM) 2.5 ** ** 2.0

1.5 cells/well) 4 1.0 + Erastin (10 M)

Cell number Promethazin Rifampicin

(1 x 10 x (1 0.5

0

+ Erastin (10 M)

Supplemental Figure 4. Anti-ferroptotic drugs did not rescue inhibition of cell proliferation by high-dose erastin treatment Cell number and images of MDA-MB-231 cells treated with indicated compounds for 48 h. Erastin (10 M), promethazine (1 M), and rifampicin (10 M). n=3 **P<0.01 (ANOVA) Supplemental Figure 5

A B Cis (-)

1.0 Cis + PMZ (n) 0 h Day 4 Day 14

0.8 ** (4) Cisplatin (17 mg/kg i.p) 0.6 Cis + RIF (8)

+ PMZ (every 12h) 0.4 =0.3 P

Survival rate Cis (8) 0.2 + RIF (every 12h) (8) 0 02468101214 Days

Supplemental figure 5. Effects of anti-ferroptotic drugs on survival rateincisplatin- induced AKI mice A. Experimental scheme. Promethazine (PMZ, 20 mg/kg) and rifampicin (RIF, 20mg/kg) were orally administered every 12 h for 4 days from 30 min prior to the cisplatin (17 mg/kg i.p.) injection, and they were observed daily for 14 days. B. Survival rate after cisplatin injection. **P<0.01 (log-rank test) Supplemental Figure 6

A B P=0.07 (n) 350 P=0.06 Cisplatin (-) 1.5 (3) 300 250 Cisplatin (17 mg/kg) + DMSO 200 1.0 (9) 150

+ Fer-1 (20 mg/kg) BUN (mg/dL) 100 0.5 Creatinine (mg/dL) (7) 50 -30 m 5 h 96 h 0 0

+ Cisplatin + Cisplatin C Cis (-) Cis + DMSO Cis + Fer-1 Masson trichrome

Supplemental figure 6. Effects of Fer-1 on cisplatin-induced AKI A. Experimental scheme. Fer-1 (20 mg/kg) were intraperitoneally administered 30 min prior to and at 5 h after the cisplatin (17 mg/kg) injection. B. The levels of BUN and plasma creatinine. P valueareshown(t-test). Data are mean ± S.E.M. C. Histological images of kidneys of Masson trichrome staining. Scale, 100 m. Supplemental Figure 7

A LLC-PK1 DMSO RSL3 (30 nM) PMZ RSL3 + PMZ 1.2 Fer-1 1.2 RSL3 + Fer-1 1.0 1.0 1.2 * 0.8 0.8 1.0 * * 0.6 0.6 0.8 0.4 0.4 0.6 0.4 0.2 0.2 0.2 Cell viability (relative) Cell viability

0 0 (relative) Cell viability 0 01210 01210 Cisplatin (μg/mL) Cisplatin (μg/mL) Cisplatin Cisplatin + (2 μg/mL) RSL3 (30 nM) B NRK52E DMSO RSL3 (0.3 μM) PMZ RSL3 + PMZ 1.2 Fer-1 1.2 RSL3 + Fer-1 1.0 1.0 1.5 * 0.8 0.8 * * 0.6 0.6 1.0 0.4 0.4 0.5 0.2 0.2 Cell viability (relative) Cell viability 0 0 012100 01210 (relative) Cell viability Cisplatin (μg/mL) Cisplatin (μg/mL)

Cisplatin Cisplatin + (2 μg/mL) RSL3 (0.3 μM) RSL3 (30 nM) Cis Cis C Cis (-) (2 μg/mL) Cis (-) (2 μg/mL) D Mouse kidney DMSO Cis (-) Cis (+)

Gpx4 17 kD

PMZ -actin

Fer-1

Supplemental Figure 7. Protective effect of anti-ferroptotic drugs on cell damage by cisplatin plus GPX4 inhibition A, B. Cell viability of cisplatin-treated renal tubule-derived cells incubated with or without RSL3. PMZ, promethazine (1 μM); Fer-1, ferrostatin-1 (1 μM). LLC-PK1 cells (in A) were assayed in DMEM medium containing 1.0 g/L glucose and 1% FBS. NRK52E cells (in B) were assayed in DMEM medium containing 4.5 g/L glucose and 5% FBS. Cell viability was measured at 48 h after the addition of cisplatin. n=3. *P<0.05 (ANOVA). Data are mean ± S.E.M. C. Cell images of LLC-PK1 cells. Scale, 50 m. D. Gpx4 expression in the mouse kidney treated with vehicle (Cis[-]) or cisplatin (Cis[+]). The kidney was collected 4 days after the cisplatin (17 mg/kg, i.p.) or vehicle injection. Supplemental Figure 8

Glucose 1.0 g/L, FBS 1% Glucose 1.0 g/L, FBS 1% Glucose 4.5 g/L, FBS 10% Glucose 4.5 g/L, FBS 10% 1.4 1.2 1.2 * * 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 * 0.2 0.2 Cell viability (fold Cell viability change) 0 (fold Cell viability change) 0 0.05 0.1 0.2 0.5 1 2 5 0.003 0.005 0.01 0.02 0.03 0.1 Erastin [M] RSL3 [M]

Supplemental figure 8. Effect of medium condition on ferroptosis induction by erastin and RSL3 H9C2 cells were treated with erastin or RSL3 for 24 h in the indicated medium. n=3. *P<0.05 vs glucose 1.0 g/L, FBS 1% group. Detailed methods and materials Materials For cell experiments, each compound was prepared as stock solution in dimethyl sulfoxide (DMSO) except BSO that are dissolved in water, and stored at -30°C.

Cell lines H9C2, NRK49F, HK2, C2C12, MDA-MB-231, NRK52E, and LLC-PK1 cells were obtained from ATCC. Panc-1 cells were obtained from Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). HT-22 cells were purchased from Millipore. Human urine-derived podocyte-like cells (HUPEC) were provided from Dr. Jeffrey B. Kopp (NIH, Bethesda).1 All cell lines except HUPEC were maintained in DMEM high glucose (4.5 g glucose/L) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37 °C with 5% CO2. HUPEC was maintained in RPMI 1640 supplemented with 10% FBS and 1% insulin, transferrin, selenium solution (Thermo) at 33 °C, and incubated at 37 °C for in the experiments.

Ferroptosis induction using different medium variants H9C2 (4,000 cells per well) were plated in 96-well plates and cultured in high glucose DMEM (4.5 g glucose/ L) supplemented with 10% FBS, high glucose DMEM (4.5 g glucose/L) supplemented with 1% FBS, or low glucose DMEM (1.0 g glucose/ L) supplemented with 1% FBS overnight. On the next day, cells were treated with a dilution series of BSO. Cell viability, released LDH, and cell images were assessed after 60 h incubation. After cells were plated once, the medium was not changed until the assessment. Cell viability was measured by WST-8 assay using Cell Count Reagent SF (Nacalai tesque). This is a colorimetric assay for the determination of the number of viable cells in cell proliferation and cytotoxicity assays by the measurement of the absorbance at 450 nm based on the reduction of a water-soluble tetrazolium salt (WST-8) by cellular dehydrogenases. The cell viability was expressed as relative values compared to the control sample, which was defined as 1.0. Released LDH was evaluated using LDH Cytotoxity Detection kit (Takara Bio).

Screening of anti-ferroptotic compounds H9C2 cells plated in 96-well plate with DMEM (1.0 g glucose/ L, 1% FBS) were cotreated with 100 M BSO and a dilution series of the compounds listed in Supplemental table 1. After 60 h incubation, cell viability was measured and expressed as relative values in relation to that measured in the non-BSO treated sample defined as 1.0. When the mean relative value of the cell viability was more than 0.5 at any one of the screened concentrations of the compound, the compound was considered as having anti-ferroptotic activity.

Induction of ferroptosis and cell death For the following cell experiments, low glucose DMEM (1.0 g glucose/ L) supplemented with 1% FBS was used as culture medium after seeding on 96-well plate unless otherwise noted. H9C2 (4,000 cells per well), HK-2 (3,000 cells), HUPEC (3,000 cells), C2C12 (2,500 cells), NRK49F (3,000 cells), Panc-1 (2,000 cells), MDA-MB-231 (2,000 cells), NRK52E (2,500 cells), LLLC-PK1 (2,500 cells) were seeded in 96-well plates 1 and allowed to adhere overnight. For BSO experiments, BSO and the compounds were added to the assay medium simultaneously, and then cell viability was measured after 60 h incubation. For the other cell death experiments, cells were pretreated with the compounds 1 h prior to treatment with cell death inducers in 1.0 g glucose/ L DMEM medium containing 1% FBS, and then cell viability was measured after 24 h (erastin, RSL3, FINO2, staurosporine and menadione), 48 h (FIN56, 17AAG, UV, and methylglyoxal), or 60 h (cisplatin) incubation.

Mitochondrial imaging Mitotracker Green FM (Thermo Scientific) and dihydrorhodamine 123 (DHR 123, Wako) were used for evaluation of mitochondrial morphology and mitochondrial ROS, respectively, in H9C2 cells. For mitotracker Green staining, the cells were plated onto black, clear-bottom µClear 96-well culture plates (Greiner) and incubated in DMEM (4.5 g/L glucose and 10% FBS) for overnight. On the next day, the cells were pretreated with the compounds from 1 h before erastin treatment. After 20 h incubation with 1.5 μM erastin, the cells were stained with 50 nM of Mitotracker Green FM in DMEM (4.5 g/L glucose) without FBS. After 20 min incubation at 37 °C, the cells were washed in Hank's balanced salt solution (HBSS) and were observed in phenol red-free DMEM (1.0 g/L glucose) supplemented 10% FBS using a BZ-X810 fluorescence microscope (Keyence). For DHR123 staining, the cells were plated onto black, clear-bottom µClear 96-well culture plates (Greiner) and incubated in DMEM (1.0 g/L glucose and 1% FBS) for overnight. On the next day, the cells were treated with 100 μM BSO and the compounds. After 36 h incubation, the cells were stained with 5 μM of DHR123 in DMEM (1.0 g/L glucose) without FBS. After 20 min incubation at 37 °C, the cells were washed in Hank's balanced salt solution (HBSS) and were observed in phenol red-free DMEM (1.0 g/L glucose) supplemented 1% FBS using a fluorescence microscope.

MDA measurements MDA in the medium was measured using the TBARS Fluometric Microplate Assay (Oxford Biochemical Research). After 60 h incubation with 100 μM BSO treatment and the indicated compounds in a 96-well culture plate, the culture supernatant of H9C2 cells was used for analysis.

Imaging of lipid hydroperoxides Cellular lipid hydroperoxides were detected using the fluorescent probe Liperfluo (Dojindo). H9C2 cells were plated onto black, clear-bottom µClear 96-well culture plates (Greiner) and incubated in DMEM (1.0 g/L glucose and 1% FBS) overnight. The cells were cotreated with 100 μM BSO and the indicated compounds for 40 h. After the treatment, the medium was replaced with HBSS containing 2 μM of Liperfluo. After 30 min incubation at 37 °C, the cells were washed with HBSS and observed using a BZ-X800 fluorescence microscope (Keyence).

Glutathione quantification The GSH-Glo Glutathione Assay (Promega) was used to quantify total glutathione in cultured cells. H9C2 cells were seeded in white-walled 96 well plates at a density of 3000 cells/well in DMEM (1.0 g/L glucose) 2 supplemented with 1% FBS before analysis. From 12 h later, cells were treated with 100 μM BSO and the indicated compounds for 24 h or 1 μM erastin and the indicated compounds for 12 h until glutathione was quantified using the kit.

Lipoxygenase inhibitor assay The determination of LOX inhibitory ability was performed in 96-well plates with arachidonic acid and purified 15-LOX from soybean using a lipoxygenase inhibitor screening kit (Cayman) according to the manufacturer's protocol.

Ferrous oxidation/ xylenol orange (FOX) assay 2 FOX assay was performed as reported in the literature . FeCl3 (2 mM) in water was incubated with an equal volume of water containing 2 mM of the compounds at room temperature. After 30 min incubation, the reaction was started by adding at a final concentration of 100 μM FeCl3 and 100 μM of each compound into the FOX reagent solution containing 100 μM xylenol orange, 90% v/v methanol, 25 mM H2SO4. The absorbance at 560 nm, which shows the formation of the Fe3+/xylenol orange complex, was measured at 10 min.

Ferroptosis in iron-loaded condition H9C2 (4,000 cells per well) cells were plated in 96-well plates and cultured in DMEM (1.0 g/L glucose) supplemented with 1% FBS overnight. The cells were treated with the indicated compounds with no BSO, 200

μM BSO, or 200 μM BSO with 100 μM Fe(NH4)2(SO4)2. Cell viability was assessed after 60 h incubation. siRNA For the knockdown of Adrb1 expression, Silencer Select Pre-designed siRNA (Thermo, s236469) was used. The scrambled RNAi oligo was used as a negative control. Forty-eight hours after the transfection of siRNA into H9C2 cells using Lipofectamine RNAiMAX (Thermo) in 6-well plate, the cells were reseeded on 96-well plate and performed the subsequent analysis. The knockdown efficiency was confirmed by qPCR.

Generation of Ahr deficient cells Ahr deficient H9C2 cells were generated with the CRISPR/Cas9 system using the sgRNA CRISPR/Cas9 All- In-One set (K6920005, Applied Biological Materials). Cells were transiently transfected with the all-in-one plasmid vector inserted the knockout target sequence by Lipofectamine 2000 (Thermo) according to the manufacturer’s protocol, and selected by culture in medium containing 1 g/mL puromycin for 48 h. Subsequently, single clone cells were established. Deficient of AhR expression in the established clones was confirmed by western blotting. qPCR Total RNA of the H9C2 cells was extracted using RNeasy Mini Kit (Qiagen), and transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Quantitative PCR was performed using THUNDERBIRD Probe qPCR Mix (Toyobo) and the StepOne plus (Thermo). The following Taqman 3 probes (Thermo) were used: Cyp1a1 (Rn00487218_m1) and Actb (Rn00667869_m1). The values obtained were ΔΔCt method of relative quantification.

Free radicals trapping analysis Scavenging potentials of the compounds to superoxide and hydroxyl radical were assessed by ESR-spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).3, 4 Samples were dissolved in acetone except for ascorbate and T3 that were dissolved in water and 0.1 M NaOH, respectively. In the xanthine oxidase (XOD) system for determination of superoxide, a reaction mixture containing 50 L of 2 mM hypoxanthine, 30 L of DMSO, 50 L of the sample solution, 20 L of 4.45 M DMPO dissolved in pure water and 50 L of 0.4 U/mL XOD in pure water was prepared in a test tube. The mixtures were transferred to an ESR spectrometry cell, and the DMPO-OOH spin adduct was quantified 97 s after the addition of XOD. The measurement conditions of the ESR (JES-FA-100, JEOL, Tokyo) were as follows: field sweep, 330.5_340.5 mT; field modulation frequency, 100 kHz; field modulation width, 0.07 mT; amplitude, 200; sweep time, 2 min; time constant, 0.1 s; microwave frequency, 9.42 GHz; microwave power, 4 mW. The concentrations of the DMPO-OOH were determined from the peak area of the first signal of each spin adducts. For ESR analysis of the hydroxyl radical from the Fenton reaction, 50 L of 2 mM hydrogen peroxide dissolved in pure water, 50 L of 89 mM DMPO dissolved in pure water, 50 L of sample, and 50 L of 0.2 mM FeSO4 dissolved in pure water were placed in a test tube and mixed. Each mixture was transferred to an

ESR spectrometry cell, and the DMPO–OH spin adduct was quantified 113 s after the addition of FeSO4. The measurement conditions for ESR were as follows: field sweep, 330.5–340.5 mT; field modulation frequency, 100 kHz; field modulation width, 0.1 mT; amplitude, 25; sweep time, 2 min; time constant, 0.1 s; microwave frequency, 9.42 GHz; microwave power, 4 mW. The concentrations of the DMPO-OH were determined from the peak area of the second signal of each spin adducts. Scavenging potential of the compounds to AAPH-derived peroxyl radical was assessed using OxiSelect ORAC Activity Assay Kit (Cell Biolabs). Effects of compounds (1, 3, 10, and 100 M) were calculated as Trolox equivalent.

Lipid peroxyl radical scavenging analysis Lipid peroxyl radicals generated in an AA/LOX system were detected with a fluorescence probe NBD-Pen.5 In this system, arachidonic acids (>95.0%, Sigma-Aldrich) and LOX from Glycine max soybeans (Sigma- Aldrich) were used. A mixture of 160 L phosphate buffered saline pH 7.4 (PBS) containing AA (final 500 M) and the indicated final concentration of compounds were prepared in a black-walled 96 well palate. A solution of 20 L NBD-Pen (final 5 M) and 20 L of LOX solution (final 10 g/mL) was added to the mixture, and the fluorescence intensity (wavelengths: excitation, 470 nm; emission, 530 nm) was measured at 37 °C using a Spectra Max M2e (Molecular Devices). For the NBD-Pen experiments using cell extracts, 150,000 H9C2 cells per well seeded on 6-well plates were incubated with DMSO or drugs in DMEM (1.0 g/L glucose) supplemented with 1% FBS for 12 h, and then the cells were collected by trypsin/EDTA. The collected cell pellets were added 50 L of 100% methanol and sonicated for 1 min. After centrifugation at 4 °C and 12,000 g for 5 m, deproteinized cell extracts 4 in the aqueous phase was obtained. The 20 L aliquots of the extracts were used for the experiment with NBD- Pen in the AA/LOX system (total 200 L per sample) as described above.

PCOOH experiments PCOOH was enzymatically synthesized from 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0- 18:2 PC, purchased from Avanti Polar Lipids) using soybean lipoxygenase-1 and chromatographically purified.6, 7 The predominant sn-2 residue of the purified PCOOH was 13-hydroperoxyoctadecadienoic acid. For in vitro detection of PCOOH-derived PCOO • by NBD-Pen, a mixture of 190 μL PBS containing PCOOH (final 0, 15, 50 μM) and NBD-Pen (final 5 μM) was prepared in a black-walled 96-well palate. A 10 μL solution of Fe(NH4)2(SO4)2 (final 50 μM) or water was added to the mixture, and the fluorescence intensity was measured as described above. To detect of cellular lipid-derived radicals, H9C2 cells (4,000 cells/well) were plated onto black, clear-bottom µClear 96-well culture plates (Greiner) and incubated in DMEM (1.0 g/L glucose and 1% FBS) overnight. Culture medium was discarded and phenol red-free DMEM with 1% FBS and NBD-Pen (1 μM) were added. After 10 min, the cells were rinsed twice with PBS and then incubated with phenol red-free DMEM with 1% FBS with or without PCOOH (50 μM) for 1.5 h at 37 °C. Hoechst33342 (1 μg/ml, Dojindo) staining was performed 1 h before the addition of NBD-Pen. Fluorescence imaging was conducted with a BZ- X800 fluorescence microscope (Keyence). GFP filter cube (ex 470/40 nm, em 525/50 nm, dichroic mirror 495 nm) was used for detection of NBD-Pen signals. For PCOOH-loaded cell experiments, H9C2 cells (4,000 cells per well) were plated in 96-well plates and cultured in DMEM (1.0 g/L glucose) supplemented with 1% FBS overnight. The cells were treated with the indicated concentration of the drugs and 100 μM BSO, and 1 h later PCOOH was added into the medium. Cell viability was assessed after incubation at the indicated times.

Glutamate-induced cytotoxicity HT22 cells (2,000 cells per well) were seeded in DMEM medium containing 1.0 g/L glucose and 1% FBS onto 96-well plate for overnight. The cells were treated with 4 mM glutamate and, cell viability and released LDH activity was measured after 24 h incubation.

Animal experiments All animal experiments were approved by the Animal Committee of Tohoku University, School of Medicine (approved No. 2016-008-5, 2016-009-2, 2017-014-1, and 2019BeA-012). C57BL/6N male mice, aged 8–9 weeks, were used. The acute kidney injury was induced by an intraperitoneal injection of 0.5 mg/mL cisplatin solution (16 or 17 mg/kg as indicated, Nichi-Iko Pharmaceutical)8. Mice were orally treated with water only, promethazine (20 mg/kg in water) or rifampicin (20 mg/kg in 0.5% methylcellulose) every 12 h for 4 days from 30 min prior to the cisplatin injection (Figure 6A), or orally treated with promethazine (20 mg/kg) in the following groups (Figure 6F): i) no promethazine, ii) pretreatment 30 min prior to cisplatin injection, iii) treatment from 30 min prior to injection to 24 h after injection, iv) treatment from 24 to 96 h after injection, and v) treatment every 12 h from 30 min prior to injection to 96 h after injection.. They were sacrificed 4 days 5 after the cisplatin injection. In the experiment of supplemental figure 6, Fer-1 (20 mg/kg in DMSO) or vehicle were intraperitoneally administrated 30 min prior to and at 5 h after the cisplatin (17 mg/kg) injection. The acute hepatic injury was induced by intraperitoneal injection of LPS (5 μg/kg) and D-GaIN (500 mg/kg) dissolved in PBS.9 Mice were orally administered 0.5% MC, promethazine (20 mg/kg in MC) 12 h and 2 h prior to the LPS/D-GalN injection and sacrificed at 6 h after the injection. At the end of each study, the mice were euthanized by isoflurane, and samples were collected. Blood urea nitrogen and creatinine were assessed using a blood analyzer (i-STAT, Abbott). Plasma AST was measured by Colorimetric Analyzer (DRI-CHEM 7000V, Fujifilm).

Histology and immunohistochemistry Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin/eosin and Masson’s trichrome.10 For immunohistochemistry, sections were immunolabeled using antibodies for anti-HEL (JaICA, clone 5F12), anti-4 HNE (JaICA, clone HNEJ-2), and anti-KIM-1 (R&D, AF1817). For anti-KIM-1 staining, deparaffinized sections were heated for antigens retrieval for 5 min at 120 °C in 0.01 M citrate buffer pH 6.0, and then incubated with the anti-KIM-1 antibody (1:400) overnight at

4 °C, followed by blocking with 3% H2O2 in methanol and incubation with horseradish peroxidase–labeled anti-goat IgG (Histofine Simple Stain Max PO goat; Nichirei)11. The slides were visualized using 3,3’- diaminobenzidine (DAB) and counterstained with Mayer’s Hematoxylin. For anti-HEL and 4-HNE staining, deparaffinized sections (for anti-HEL) or antigen retrieved-sections by heating for 5 min at 120 °C in 0.01 M citrate buffer pH 6.0 (for anti-4 HNE) were incubated with 10% normal rabbit serum for 20 min at room temperature, and then incubated with anti-HEL (1:50) or anti-4 HNE antibody (1:10) overnight at 4 °C. The sections were treated with a streptavidin biotin complex alkaline phosphatase kit (Histofine SAB-AP [M] kit; Nichirei) and Vector Red Substrate kit (Vector Laboratories) for visualization. TUNEL staining was performed using the ApopTag peroxidase in situ apoptosis detection kit (Millipore) in a Ventana Discovery XT Immunostainer (Roche). The number of TUNEL positive cells were counted per ×200 high power field. Visualization of mitochondria in the kidney was performed by a fluorescence observation of a formalin fixed paraffin embedded section stained with Masson’s trichrome.12 The section was observed using a BZ-X800 fluorescence microscope (Keyence) equipped with a 100× objective lens and Texas-Red filter cube (ex 560/40 nm, em 630/75 nm, dichroic mirror 585 nm).

Western blotting Mouse kidney were homogenized in RIPA lysis buffer (Santa Cruz) and centrifuged 15,000 × g at 4 °C for 15 min. The supernatant was collected and used as the protein sample. Western blotting was performed by standard immunoblotting procedure with anti-GPX4 antibody (1:1000, Abcam #125066) and Anti-β-Actin pAb-HRP-DirecT (1:5000, MBL#PM053-7).

Quantification and statistical analysis Statistical information for individual experiments can be found in the corresponding figure legends. Values are presented as mean ± S.E.M. Statistical analyses were conducted using JMP 14 software (SAS Institute). 6

Statistical comparisons between groups were analyzed for significance by two-tailed Student’s t-test or one- way analysis of variance (ANOVA) with Dunnett’s or Bonferroni post hoc test. Results were considered significant at P values of <0.05.

References for detailed method section 1. Sakairi T, Abe Y, Kajiyama H, Bartlett LD, Howard LV, Jat PS, et al.: Conditionally immortalized human podocyte cell lines established from urine. Am J Physiol Renal Physiol, 298: F557-567, 2010 2. Tadolini B, Franconi F: Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism. Free Radic Res, 29: 377-387, 1998 3. Niwano Y, Sato E, Kohno M, Matsuyama Y, Kim D, Oda T: Antioxidant properties of aqueous extracts from red tide plankton cultures. Biosci Biotechnol Biochem, 71: 1145-1153, 2007 4. Sato E, Mokudai T, Niwano Y, Kohno M: Kinetic analysis of reactive oxygen species generated by the in vitro reconstituted NADPH oxidase and xanthine oxidase systems. J Biochem, 150: 173-181, 2011 5. Yamada K, Mito F, Matsuoka Y, Ide S, Shikimachi K, Fujiki A, et al.: Fluorescence probes to detect lipid- derived radicals. Nat Chem Biol, 12: 608-613, 2016 6. Kato S, Nakagawa K, Suzuki Y, Suzuki K, Mizuochi S, Miyazawa T: Preparation of 13 or 9-hydroperoxy- 9Z,11E (9E,11E) or 10E,12Z (10E,12E)-octadecadienoic phosphatidylcholine hydroperoxide. J Oleo Sci, 63: 431-437, 2014 7. Suzuki Y, Nakagawa K, Kato S, Tatewaki N, Mizuochi S, Ito J, et al.: Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells. Biochem Biophys Res Commun, 458: 920-927, 2015 8. Mishima E, Inoue C, Saigusa D, Inoue R, Ito K, Suzuki Y, et al.: Conformational change in transfer RNA is an early indicator of acute cellular damage. J Am Soc Nephrol, 25: 2316-2326, 2014 9. Shima H, Sasaki K, Suzuki T, Mukawa C, Obara T, Oba Y, et al.: A novel indole compound MA-35 attenuates renal fibrosis by inhibiting both TNF-alpha and TGF-beta1 pathways. Sci Rep, 7: 1884, 2017 10. Mishima E, Fukuda S, Shima H, Hirayama A, Akiyama Y, Takeuchi Y, et al.: Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD. J Am Soc Nephrol, 26: 1787-1794, 2015 11. Suzuki T, Yamaguchi H, Kikusato M, Hashizume O, Nagatoishi S, Matsuo A, et al.: Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage. J Am Soc Nephrol, 27: 1925-1932, 2016 12. Matsumoto A, Sakaguchi Y, Namba T, Mizui M, Hamano T, Isaka Y, et al.: Elastica Masson's Trichrome (EMT) Staining Is Useful for the Visualization of Podocyte Foot Process and Tubular Mitochondria in the Kidney. Presented at the American Society of Nephrology (ASN) Kidney Week 2017 Annual Meeting; November 3, 2017; New Orleans, LA, 2017

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