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Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed of the Collecting Duct

Huihui Huang,1,2 William W. Jin,1 Ming Huang,1 Heyu Ji,1 Diane E. Capen,1 Yin Xia,3 Junying Yuan,4 Teodor G. Paunescu,˘ 1,2 and Hua A. Jenny Lu1,2

1Center for Systems , Program in Membrane Biology and Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 2Harvard Medical School, Boston, Massachusetts 3Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China 4Department of Cell Biology, , Boston, Massachusetts

ABSTRACT Background Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Methods Mice treated with gentamicin for 7 days developed AKI, and programmed pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Results Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with , occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine- protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addi- tion, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflam- mation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, 2 2 deletion of RIPK3 in the Ripk3 / mice significantly attenuated gentamicin-induced AKI. Conclusions A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin- induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

JASN 31: 2097–2115, 2020. doi: https://doi.org/10.1681/ASN.2019020204

Received February 26, 2019. Accepted May 11, 2020. Aminoglycosides, such as gentamicin, are one of Published online ahead of print. Publication date available at the most commonly used classes of antibiotics to www.jasn.org. treat gram-negative bacterial infection. However, its clinical application has been greatly limited by Correspondence: Dr. Hua A. Jenny Lu, Center for Systems Bi- ology, Program in Membrane Biology and Division of Nephrol- its associated nephrotoxicity. The incidence of ogy, Department of Medicine, Massachusetts General Hospital, gentamicin-induced AKI ranges from 2% to as 185 Cambridge Street, CPZN 8150, Boston, MA 02114. Email: high as 55%.1 Itisestimatedthatupto30%of [email protected] patients treated with gentamicin for more than Copyright © 2020 by the American Society of Nephrology

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7 days exhibit signs of renal impairment.2 Multiple efforts to Significance Statement prevent gentamicin-induced AKI have not been effective due to the lack of understanding of the specific mechanism un- Gentamicin-induced AKI is a commonly recognized clinical prob- derlying gentamicin-induced tubular injury. It was observed lem, but the mechanism is not well understood. A mouse model of a long time ago that gentamicin was primarily taken up by gentamicin-induced AKI revealed a previously unrecognized role of necroptosis in mediating collecting duct epithelial cell death, in- and accumulated in proximal tubular cells and caused the terstitial inflammation, and fibrosis. Importantly, either inhibiting a proximal tubular injury. For many years, it was believed necroptotic pathway activator RIPK1 kinase with its inhibitor Nec-1 that the main mechanism of gentamicin-induced AKI is tu- or deleting a key necroptotic gene, Ripk3,significantly attenuated bular cell apoptosis, which occurs predominantly in proxi- gentamicin-induced AKI in mice and in cultured porcine and murine fi mal tubules in experimental animals and patients treated kidney tubular cells. Identi cation of a novel programmed nec- roptosis pathway in gentamicin-induced renal tubule injury could 3,4 with gentamicin. However, treatment to inhibit apoptosis provide a new therapeutic target. using various methods only partially improves gentamicin- induced AKI in animals.5–7 Additional and/or alternative tu- bular injury mechanisms underlying gentamicin-induced becomes oligomerized and then translocates from the cytosol AKI are under active investigation.8 to the plasma membrane where it disrupts the latter.19 In Very interestingly, unlike other types of acute tubular injury addition, a very recent study has demonstrated the existence resulting in oliguric AKI, gentamicin-induced AKI is frequently of a repair mechanism mediated by the endosomal sorting associated with polyuria and reduced urine osmolality, which complex required for transport (ESCRT-III). Through shed- implicates a possible defect of the urinary concentration mech- ding of MLKL-damaged membrane, ESCRT-III is able to anism, a main function of the collecting duct (CD). Whether sustain cell survival despite MLKL activation.20 When cells gentamicin-induced AKI could involve other type(s) of tubular undergo necroptosis, they present with organelle and cell cell injury mechanisms and/or other tubular segments, such as swelling, permeabilization of the plasma membrane, and CDs, besides proximal tubules is not clear. Using an anti- spilling of intracellular contents.21,22 Emerging studies have gentamicin antibody to stain gentamicin-treated mouse kid- revealed that blocking necroptosis using a RIPK1 inhibitor or ney, it was found that, besides a predominant accumulation of RIPK3-deficient mice largely alleviated multiple kidney in- gentamicin in proximal tubular cells, there was a significant juries induced by ischemia reperfusion; chemical injury accumulation of gentamicin in the CDs. Those CD cells that from cisplatin, cyclosporine, and contrast dye; and by unilat- – accumulated gentamicin appeared “round” or swollen with eral ureteral obstruction.23 25 prominent nuclei.9 However, how the CD tubular cells took It has been well appreciated that gentamicin causes signif- up gentamicin and whether they contributed to gentamicin- icant AKI and some of the patients with gentamicin-induced induced AKI remains unknown. This study suggests that AKI progress to ESKD. Massive inflammation and tubular uptake of gentamicin by CD leads to CD necroptosis. CD damage were reported in animals and patients treated with necroptosis likely contributes critically to the development of gentamicin.26 Whether necroptosis, in addition to apoptosis, is massive inflammation and interstitial fibrosis in gentamicin- involved in gentamicin-induced tubular injury has not been elu- induced AKI. cidated. To understand the pathologic mechanism underlying Several types of cell death, including apoptosis and necro- gentamicin-induced AKI, we performed a study to re-examine sis, occur during kidney tubular injury. Apoptosis is a regu- the tubular cell injury in gentamicin-treated mice. We demon- lated cell death that has been well studied for decades in many strated the presence of widespread interstitial inflammation, fi- tissues, including the kidney.10,11 Tubular cell apoptosis was brosis, and significant renal dysfunction after 7 days of treatment once considered to be a key form of cell death leading to CKD. with gentamicin in mice. Besides a low grade of apoptosis oc- However, this point of view was recently challenged given the curring in proximal tubules, we have unexpectedly detected discovery of a regulated necrosis pathway, necroptosis. Nec- widespread necroptosis in principal cells (PCs) of the CDs in roptosis is the most characterized pathway of regulated ne- gentamicin-treated animals. We further show that gentamicin crosis in higher eukaryotic cells.12,13 It is implicated in a wide induced upregulation of necroptotic signals, and inhibiting nec- range of high-grade tissue injuries such as myocardial ische- roptosis by RIPK1 inhibitor necrostatin-1 (Nec-1) or RIPK3 mia and reperfusion injury, amyotrophic lateral sclerosis, deficiency significantly alleviated gentamicin-induced inflam- sepsis, and intestinal inflammation.14–16 The best-studied mation, interstitial fibrosis, and kidney dysfunction. necroptotic pathway is the TNF-induced necroptosis through activation of TNF receptor 1.17,18 Induction of necroptosis is METHODS mediated through activation of TNF receptor 1 and subse- quent activation and interaction of the receptor-interacting Animals protein kinase 1 (RIPK1) and RIPK3. RIPK1 and RIPK3 in- C57/BL6J mice were purchased from The Jackson Laboratory teraction leads to recruitment and phosphorylation of the (Bar Harbor, ME). The mice were given free access to food and 2 2 mixed lineage kinase domain-like pseudokinase (MLKL), water under a 12-hour light/dark cycle. Ripk3 / mice were forming the necrosome. Phosphorylated MLKL (pMLKL) kindly provided by Dr. Vishva Dixit from Genentech.27 All

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C Relative protein expression MW (fold change over internal control) E (KDa) Ctrl Genta 4 01234 ** 25 3 NGAL (Tissue) Genta 2 37 NGAL (Tissue) ** Ctrl GAPDH 1

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Figure 1. Gentamicin causes kidney dysfunction with inflammation and renal fibrosis. Gentamicin (80 mg/kg per day) was given to C57BL/6 mice (n56) through IP injection for 7 days. On day 6, mice were transferred to metabolic cages for 24 hours. (A) Urine output was significantly increased and urine osmolality was significantly reduced in gentamicin-treated (Genta) mice compared with the control (Ctrl) group. (B) SCr and BUN were significantly increased in gentamicin-treated mice. (C) The NGAL level was significantly increased in both kidney lysate and urine of gentamicin-treated mice by immunoblotting. Images were quantified using ImageJ software (NIH) and statistical analysis was performed using t test (left panel). (D) H&E staining revealed kidney tubular injury in

JASN 31: 2097–2115, 2020 Gentamicin Causes Necroptosis in Kidney 2099 BASIC RESEARCH www.jasn.org animal experiments were conducted according to the National 10% SDS-PAGE. Gels were stained with SimplyBlue SafeStain Institutes of Health (NIH) Guide for the Care and Use of (Invitrogen, Carlsbad, CA) for 1 hour and washed with double- Laboratory Animals and were approved by the Massachusetts distilled water for 1 hour. BSA (Santa Cruz Biotechnology, General Hospital (MGH) Subcommittee on Research Animal Dallas, TX) was used for the albumin control band. Care. All mice were anesthetized using isoflurane. Mice sub- jected to perfusion were perfused through the left cardiac ven- Serum Creatinine and BUN Measurements tricle with PBS containing 0.9% sodium chloride in 10 mM Terminal blood collection was performed. Serum was sepa- phosphatebufferatpH7.4,atarateof13–17 ml/min for rated using a BD Microtainer (Becton-Dickinson, Franklin 4 minutes, followed by modified paraformaldehyde-lysine- Lakes, NJ) and stored at 280°C. Serum creatinine (SCr) levels periodate (PLP) for 4 minutes. Modified PLP consisted of were measured with the QuantiChrom Creatinine Assay Kit 4% paraformaldehyde, 75 mM lysine-hydrochloride, 10 mM (BioAssay Systems, Hayward, CA) according to the manufac- sodium periodate, and 0.15 M sucrose in 37.5 mM sodium turer’s protocol. BUN levels were measured with the Stanbio phosphate. Kidneys from perfusion-fixed mice and mice that Urea Nitrogen Kit (Stanbio Laboratory, Boerne, TX) accord- were not perfused were fixed with modified PLP at 4°C over- ing to the manufacturer’s protocol. night, washed with PBS for 333 hours, and stored in PBS containing 0.02% sodium azide until use. Hematoxylin and Eosin Staining Nonperfused, modified PLP-fixed kidney tissues were embed- Experimental Groups and Drug Treatments ded in paraffin blocks and sectioned at 5-mm thickness. Sec- C57/BL6J mice (8 weeks old) were injected intraperitoneally tions were used for hematoxylin and eosin (H&E) staining (IP) with gentamicin (Thermo Fisher Scientific, Waltham, according to the manufacturer’sprotocol.Briefly, sections MA) at 80 mg/kg per day for 7 days to induce kidney injury. were deparaffinized with xylene, rehydrated in serial dilutions For the Nec-1 (Sigma-Aldrich, St. Louis, MO) experiment, of ethanol, and stained with the Weigert iron hematoxylin set mice were divided into three groups. The first group was the (Sigma-Aldrich) for staining nuclei. Sections were rinsed with control and was given vehicle (DMSO, 1.65 mg/kg per day) normal tap water, differentiated with 0.3% acid alcohol, and through IP injection for 7 days. The second group was pretreated then “blued” with Scott’s tap water (238 mM sodium bicar- withvehicle(DMSO,1.65mg/kgperday)30minutesbeforeIP bonate and 29 mM magnesium sulfate in distilled water) for gentamicin injection at 80 mg/kg per day for 7 days. The third 2 minutes. Sections were then treated with 1% eosin (Sigma- group was pretreated with Nec-1 (1.65 mg/kg per day) 30 minutes Aldrich) for cytosol staining, dehydrated, and mounted. before IP gentamicin injection at 80 mg/kg per day for 7 days. Picrosirius Red Staining and Masson Trichrome Staining Metabolic Cage Studies Staining was performed with the Picrosirius Red Stain Kit (Poly- C57/BL6J mice (8 weeks old) in the control and gentamicin sciences Inc, Warrington, PA) or Trichrome Stain Kit (Masson, treatment groups were subjected to 24-hour metabolic cage Sigma-Aldrich) according to the manufacturer’s protocol. The monitoring. Urine samples were collected and water and food sections were examined with a Zeiss LSM800 confocal micro- intake was recorded. Urine osmolality was measured with a scope (Carl Zeiss Microscopy, Thornwood, NY) under normal Vapor Pressure Osmometer 5520 (Wescor, Logan, UT). and polarized light. Analysis of staining intensity was performed using ImageJ software (NIH, Bethesda, MD). Urinary Analysis Urinaryanalysiswasperformedaspreviouslydescribed.28 Immunofluorescence Staining and Immunoblotting Spot urine samples (2 ml) from each group were mixed Perfused, modified PLP-fixed kidney tissues were embedded with 5 ml of SDS sample loading buffer, and subjected to in ornithine carbamyl transferase or paraffin and sectioned gentamicin-treated mice. Brush border disruption, tubular dilation, tubular cell injury, and cast formation were observed in gentamicin- treated mice. Scale bar, 20 mm. (E) Kidney injury score was significantly increased in gentamicin-treated mice. Kidney injury score was calculated based on displayed tubular necrosis, cast formation, and tubular dilation as follows: 0, normal; 1, ,10%; 2, 10%–25%; 3, 26%–50%; 4, 51%–75%; 5, .75%. (F) Picrosirius Red staining for collagen fibrils revealed significantly increased collagen deposition in gentamicin-treated mouse kidney. Picrosirius red staining was viewed under normal light (bright field, upper left panel) and polarized light (lower left panel). Scale bar, 20 mm. Immunofluorescence staining of gentamicin-treated mouse kidney with antibodies against FN, a-SMA, and Col1 (right panels). All sections were costained with anti-AQP2 antibody (green) to identify CDs in the renal cortex. Blue represents 49,6-diamidino-2-phenylindole staining. (G) Markedly increased F4/80-positive macrophages and Ly6G-positive neutrophils were detected by immunofluorescence staining in gentamicin-treated mouse kidney. Scale bar, 20 mm. (H) Increased mRNA level of a-SMA, FN,andCol1 was detected in gentamicin-treated mice by quantitative real-time PCR. The mRNA level of proinflammatory cytokines including TNF-a, IL-6,andIL-1b was significantly increased by quantitative real-time PCR analysis in the gentamicin-treated group compared with the control group. Bar values represent mean6SEM, n56 replicates per group, *P,0.05,**P,0.01, ***P,0.001 versus control. Statistics was performed using t test for normal distribution, using Mann–Whitney test for non-normal distribution. MW, molecular weight.

2100 JASN JASN 31: 2097–2115, 2020 www.jasn.org BASIC RESEARCH A a B a

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PC Genta Genta IC PC

10 µm 2 µm 2 µm

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Figure 2. TEM examination of CD tubular cell injury caused by gentamicin. Ultrastructural features of necroptosis were detected in CD PCs in mouse kidney after treatment with gentamicin for 7 days. (A) CD PCs in gentamicin-treated (Genta) mice were swollen with more lucent cytoplasm (b) in contrast to adjacent ICs and CD PCs in the control (Ctrl) mice (a). (B) Apical membrane was observed in the swollen PC under low magnification (a) and high magnification (b and c). Arrows indicate ruptured plasma membrane. (C) Mild swelling of mitochondria with enlarged crista junction was observed in CD PC in gentamicin-treated mouse kidney (b), compared with adjacent IC and CD PC in control mice (a). (D) Increased infiltration of multinucleic neutrophils was observed in the interstitium (a). Deposition of extracellular matrix (arrows) was detected around injured CDs. An increased number of activated fibroblasts (arrowheads) with enriched cytoplasm and expanded processes were present around injured CDs (b and c). Scale bars were marked on each image.

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A Ctrl Genta B pMLKL pMLKL/AQP2 pMLKL/V-ATPase Merge Control MLKL

pRIPK3 pRIPK3/AQP2 pRIPK3/V-ATPase Merge AQP2 Control Genta MLKL / AQP2 Genta

C Ctrl Genta D MW 6 * (KDa) Ctrl 50 pMLKL Genta

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37 Relative protein expression Caspase3 (fold change over internal control) Cleaved 15 Caspase3 0 37 GAPDH MLKL pMLKL RIPK3 p-RIPK3 Cleaved Caspase3 E 2.5 2.5 1.5 * ** 2.0 2.0 1.0 1.5 1.5 1.0 1.0 0.5

Mlkl / β -Actin 0.5 0.5 Ripk1 / β -Actin Ripk3 / β -Actin 0 0 0 Ctrl Genta Ctrl Genta Ctrl Genta

Figure 3. Upregulation of necroptotic signaling in kidney in gentamicin-treated mice. (A) Immunofluorescence staining for MLKL in gentamicin-treated (Genta) kidney. In control (Ctrl) mice under baseline conditions, immunostaining of MLKL (red) was located mainly in cytoplasm and basal membrane in CD PCs, whereas in gentamicin-treated mice, MLKL immunostaining mainly accumulated on the apical membrane of PCs. CD PCs were identified by costaining with an anti-AQP2 antibody (green). (B) Immunofluorescence signal of pMLKL and pRIPK3 was dramatically increased and clearly accumulated on the apical plasma membrane in CD PCs in

2102 JASN JASN 31: 2097–2115, 2020 www.jasn.org BASIC RESEARCH at 5-mm thickness for immunofluorescence staining. Cryosec- Signaling Technology), anti-RIPK3 diluted 1:1000 (#sc- tions were used to detect MLKL using an anti-MLKL antibody 374639; Santa Cruz Biotechnology), and anti-pRIPK3 diluted 1:500 (#AP14272b; Abgent, San Diego, CA). The pri- (Thr231/Ser232) diluted 1:1000 (#57220). mary antibodies used for paraffin sections were anti-AQP2 diluted 1:1600 (#sc-9882; Santa Cruz Biotechnology), anti RNA Extraction and Quantitative Real-Time PCR V-ATPase (provided by Dr. Dennis Brown) diluted at 1:800, RNA was extracted from control and treated mice. Briefly, anti-fibronectin (anti-FN) diluted 1:2000 (#F3648; Sigma- mouse kidneys from each group were homogenized in TRIzol Aldrich), anti–a-smooth muscle actin (anti–a-SMA) diluted (Invitrogen) and separated using chloroform after centrifuga- 1:2000 (#A5228; Sigma-Aldrich), anti–collagen type I (anti- tion. RNA was precipitated with isopropanol, washed with Col1) diluted 1:200 (#C2456; Sigma-Aldrich), anti-F4/80 di- 70% ethanol, and dissolved in diethyl pyrocarbonate–treated luted 1:100 (#14-4801-85; Invitrogen), anti-MLKL (phosphor water. cDNA synthesis was carried out with the iScript cDNA S358) diluted 1:1500 (#ab196436; Abcam, Cambridge, United Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufac- Kingdom), anti–phosphorylated RIPK3 (anti-pRIPK3; turer’s protocol. Quantitative real-time PCR was performed using Thr231/Ser232) diluted 1:500 (#57220; Cell Signaling Tech- the QuantStudio 3 Real-Time PCR system (Thermo Fisher Sci- nology, Danvers, MA). Briefly, for cryosections, the slides were entific), using Power SYBR Green PCR Master Mix (Life Tech- rehydrated in PBS, incubated with 1% SDS for 4 minutes, and nologies, Carlsbad, CA). mRNA levels for each gene were nor- washed with PBS. For paraffin sections, the slides were depar- malized to the expression levels of the housekeeping gene b-actin. affinized and rehydrated as described above. Antigen retrieval Relative mRNA expression was determined using the Ct method. was performed by incubating slides in Tris-EDTAbuffer (10 mM The list of primer sequences is shown in Supplemental Table 1. Trizma Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) at 95°C for 20 minutes. After cooling to room temperature, the slides were Transmission Electron Microscopy washed with PBS. The slides from both cryosections and paraffin Transmission electron microscopy (TEM) was performed as sections were blocked with 1% (w/v) BSA in PBS and then in- previously described.30 Briefly, kidney slices were fixed as de- cubated with primary antibodies at 4°C overnight. After washing scribed above, rinsed in PBS, and postfixed in 2% glutaralde- with PBS, slides were incubated with fluorophore-conjugated hyde in 0.1 M sodium cacodylate buffer for 24 hours at 4°C. secondary antibodies (Jackson ImmunoResearch Laboratories, The slices were rinsed with 0.1 M sodium cacodylate buffer West Grove, PA) at room temperature for 1 hour. After washing and infiltrated with 1% osmium tetroxide in cacodylate buffer with PBS, the slides were mounted with 49,6-diamidino-2- for 1 hour at room temperature. Tissue blocks were rinsed phenylindole. Fluorescence images were acquired using a again in cacodylate buffer, fully dehydrated through a series Zeiss LSM800 confocal microscope. of graded ethanols, dehydrated briefly with 100% propylene Immunoblotting was performed as previously described oxide, and preinfiltrated with a 1:1 mix of Eponate resin (Ted using the following antibodies29:anti-FNdiluted1:1000 Pella, Redding, CA) and propylene oxide overnight on a gentle (#F3648), anti–phosphorylated-Smad3 (S423/425) diluted rotator. The following day, specimens were infiltrated with 1:1000 (#9520; Abcam), anti-Smad3 diluted 1:1000 (#9523; fresh Eponate resin, embedded in flat molds with fresh Epo- Cell Signaling Technology), anti–a–SMA diluted 1:1000 nate, and allowed to polymerize for 24–48 hours at 60°C. Thin (#A5228), anti–glyceraldehyde-3-phosphate dehydrogenase sections were cut using an EM UC7 ultramicrotome (Leica (GAPDH) diluted 1:20,000 (#2118; Cell Signaling Technol- Microsystems, Wetzlar, Germany), collected onto formvar- ogy), anti–b-actin diluted 1:10,000 (#4967; Cell Signaling coated grids, stained with uranyl acetate and lead citrate, Technology), anti-MLKL (phosphor S358) diluted 1:1500 and examined under a JEM 1011 transmission electron mi- (#ab196436), anti-MLKL diluted 1:1000 (#MABC604; croscope (JEOL, Tokyo, Japan) at 80 kV. Images were collected Millipore-Sigma, Burlington, MA), anti–neutrophil using an AMT digital imaging system (Advanced Microscopy gelatinase-associated lipocalin (anti-NGAL) diluted 1:1000 Techniques, Danvers, MA). (#AF-1857; R&D Systems, Minneapolis, MN), anti– phospho-NF-kB diluted 1:1000 (#3033; Cell Signaling Tech- Cell Culture and Cell Viability Assay nology), anti–T-NF-kB diluted 1:1000 (#8242; Cell Signaling LLC-PK1, mIMCD, and mCCDC11 cells were cultured in Technology), anti– 3 diluted 1:1000 (#9662; Cell DMEM medium (Thermo Fisher Scientific) containing 10%

gentamicin-treated kidney. Increased expression and apical membrane accumulation of pMLKL only occurred in PCs (green), but not in adjacent ICs (identified by V-ATPase staining, purple) of the same CD. (C) Protein expression of MLKL, pMLKL, RIPK3, and pRIPK3 was significantly increased in gentamicin-treated mouse kidneys compared with control mice. (D) Necroptotic protein expression was quantified using ImageJ. (E) Kidney lysates from gentamicin-treated and control mice were subjected to quantitative real-time PCR analysis. Significant increases in mRNA of Mlkl and Ripk3 were detected in gentamicin-treated mice compared with control. Ripk1 expression was not significantly different between these two groups. Bars represent mean6SEM, *P,0.05, **P,0.01 versus control. Statistical analysis was performed using t test. MW, molecular weight.

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AB0 3 2mM Genta 4mM Genta MW 0 0.2 0.5 1 6mM Genta (KDa) (mM) 2 50 MLKL *** *** 75 ### &&& RIPK3 ###&&& 1 *** ### 50 *** ### *** β-Actin *** 37

Absorbance (0D 570 nM) 0 LLC-PK1 mIMCD

CD Control 3 Genta Ctrl+ GM+ GM+N GM+N Genta+Nec-1 ### MW DMSO DMSO (30µM) (60µM) *** (KDa) 2 ### 50 MLKL *** *** *** 75 RIPK3 1 50

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EFRelative protein expression Relative protein expression (fold change over internal control) (fold change over internal control) 0 1 23 0 0.51.0 1.5 2.0

* * ** *** * MLKL MLKL &&& ## && ## 0 Ctrl * 200 * Genta ***& Genta+Nec-1 (30 µM) RIPK3 RIPK3 500 ***& 1000 # Genta+Nec-1 (60 µM)

GHCtrl Genta Genta+Nec-1 LLC-PK1 Fluorescence signal intensity /area (%) 0 5 10 15 20 MLKL/DAPI *** 10 µm ### mCCDC11 Membrane MLKL

*** pMLKL ### Ctrl

PMLKL/DAPI Genta 10 µm Genta+Nec-1

Figure 4. Gentamicin induces necroptosis in cultured renal tubular cells. (A) Gentamicin (Genta) treatment increased cell death in a dose-dependent manner in LLC-PK1 and IMCD cells. Cells were incubated with 2–6 mM gentamicin for 24 hours. Cell viability was measured by MTT assay. ***P,0.001 versus control (Ctrl), ###P,0.001 versus 2 mM gentamicin, &&&P,0.001 versus 4 mM gentamicin; n56. (B) Gentamicin induced upregulation of MLKL and RIPK3 in a dose-dependent manner in cultured cells by immunoblotting.

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FBS, in a 5% carbon dioxide humidified atmosphere at 37°C. treated mice (Figure 1C). Moreover, mild proteinuria was de- Cells were treated with gentamicin at different doses for tected in gentamicin-treated mice by Coomassie blue staining of 48 hours. Cells were harvested, lysed in lysis buffer, and sub- SDS-PAGE from spot urine (Supplemental Figure 1C). jected to immunoblotting. Cell viability was determined by H&E staining revealed the presence of dilated tubules and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium tubular atrophy in gentamicin-treated kidneys. Occasionally, (MTT) assay.31 Briefly, cultured cells were seeded onto 96-well cellular debris was found in dilated cortical tubules plates and grown to approximately 80% confluence. The cells (Figure 1D). Lower magnification of these images is shown in were then treated with different doses of gentamicin, genta- the Supplemental Figure 1A. The kidney injury score was eval- micin with vehicle, or Nec-1. At 48 hours after treatment, cells uated independently by three researchers and the score was sig- were incubated with 20 ml 5 mg/ml MTT (Affymetrix, Santa nificantly increased in gentamicin-treated mice (Figure 1E). Clara, CA) for 3 hours. After incubation, cells were treated We next examined the interstitial fibrosis in the with the MTT solvent DMSO for 5 minutes at room temper- gentamicin-treated mouse kidney. Picrosirius red staining re- ature. Absorbance was measured as OD at 570 nm on a Spec- vealed dramatically increased deposition of collagen fibrils in traMax Multi-Mode microplate reader (Molecular Devices, gentamicin-treated mouse kidney (Figure 1F, left panel). Im- San Jose, CA). munofluorescence staining with antibodies against extracellu- lar matrix including collagen type I (Col1) and fibronectin Statistical Analyses (FN), and the myofibroblast marker alpha-smooth muscle Data were analyzed using an unpaired design. For normal actin (a-SMA) showed that the expression of those proteins distribution, we used t test for two groups and one-way AN- was significantly increased in the interstitium around CDs OVA for more than two groups. For non-normal distribution, after gentamicin treatment (Figure 1F, right panel). we used a nonparametric test. To compare two groups, we It has been reported that maladaptive inflammatory re- used the Mann–Whitney test, and to compare three groups, sponse promotes interstitial fibrosis in many AKI models, the Kruskal–Wallis test. Statistical analysis was performed including animals and patients treated with gentamicin .32 with GraphPad Prism 6 (GraphPad Software, San Diego, Immunofluorescence staining using antibodies against the CA). A P value ,0.05 was considered statistically significant. macrophage marker F4/80 and the leukocyte/neutrophil Individual P values are specified in the figure legends. marker Ly6g revealed significant infiltration of macrophages and neutrophils in gentamicin-treated mouse kidney (Figure 1G). Inflammatory cells were quantified by ImageJ RESULTS (Supplemental Figure 1B). Quantitative real-time PCR re- vealed significantly increased expression of fibrogenic genes Gentamicin Causes Kidney Dysfunction, Inflammation, a-SMA, FN,andCol1, as well as of several proinflammatory and Fibrosis markers (TNF-a, IL-6,andIL-1b), in gentamicin-treated C57/B6 mice were injected with 80 mg/kg gentamicin per day mouse kidneys (Figure 1H). These results demonstrate that IP for 7 days. Metabolic parameters were obtained using met- gentamicin induces significant renal inflammation and inter- abolic cages. The urine output was significantly increased to stitial fibrosis in mice. twice the control value, and urine osmolality was reduced to half, indicating impaired urine concentration in gentamicin- Tubular Cell Injury Revealed by TEM in treated mice (Figure 1A). Renal function was assessed by mea- Gentamicin-Treated Mouse Kidney suring the level of SCr and BUN. SCr was increased from Gentamicin was reported to cause apoptosis of proximal tu- 0.2 mg/dl in the control to 0.6 mg/dl in the gentamicin- bular cells.33 However, when we examined the apoptosis of treated mice (Figure 1B, left panel). BUN was significantly ele- tubular cells in mice treated with gentamicin for 7 days by vated in gentamicin-treated mice compared with the control immunofluorescence staining, we only detected very few ap- (Figure 1B, right panel). The expression level of NGAL in both optotic cells that were stained for cleaved caspase 3, and apo- kidney and urine was significantly elevated in gentamicin- ptotic cells were barely detectable in the CD (data not shown).

Quantification of immunoblots was performed using ImageJ and illustrated in (E). *P,0.05 versus control, **P,0.01 versus control, ***P,0.001 versus control, &P,0.05 versus 0.2 mM gentamicin, &&&P,0.001 versus 0.2 mM gentamicin, ##P,0.01 versus 0.5 mM gentamicin. (C) Necroptosis inhibitor Nec-1 significantly rescued gentamicin-induced cell death in LLC-PK1 and IMCD cells as mea- sured by the MTT assay. Gentamicin (GM; 2 mM) was incubated with or without 60 mM Nec-1 (N) for 24 hours in LLC-PK1 and mIMCD cells. ***P,0.001 versus control, ###P,0.001 versus 1 mM gentamicin. (D) Nec-1 treatment prevented the upregulation of MLKL and RIPK3 expression induced by gentamicin in cells. Quantification of immunoblotting was performed using ImageJ and illustrated in graph (F). *P,0.05 versus control; #P,0.05, ##P,0.01 versus gentamicin; &&P,0.01 versus gentamicin1Nec-1 (30 mM). (G) Nec-1 treatment prevented gentamicin-induced plasma membrane translocation of MLKL in LLC-PK1 cells and membrane accumulation of pMLKL in mCCDC11 cells. (H) Fluorescence signal of MLKL and pMLKL accumulated on the plasma membrane was quantified by ImageJ and plotted. ***P,0.001 versus control, ###P,0.001 versus gentamicin. MW, molecular weight.

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A B 10 4000 # # 3000 *** 0 **2000 *** -10 1000 Urine osmolality (Mosmol/Kg H2O) -20 0 Bodyweight Change (%) Ctrl Genta Genta+Nec-1 Ctrl Genta Genta+Nec-1 CD F 4 0.8 40 * # ** *** 30 3 0.6 ### # 0.4 * 20 2 0.2 10 1 (BUN) mg/dl Kidney injury Score

0 Blood urea nitrogen 0 0 Ctrl Genta Genta Serum Creatinine (mg/dl) Ctrl Genta Genta Ctrl Genta Genta +Nec-1 +Nec-1 +Nec-1

E Ctrl Genta Genta+Nec-1 20X 40X

GHRelative protein expression (fold change over internal control) MW (KDa) Ctrl Genta Genta+Nec-1 0510 15 25 Tissue N-gal Urine * N-gal 37 GAPDH

Tissue Ctrl ** Genta Urine N-gal N-gal 20 #* Genta+Nec-1

Figure 5. Inhibition of necroptosis with Nec-1 alleviates gentamicin-induced kidney dysfunction and renal tubular injury. C57BL/6 mice were injected IP with necroptosis inhibitor Nec-1 (1.65 mg/kg per day) or vehicle (DMSO) 30 minutes before gentamicin injection daily for 7 days. (A and B) Nec-1 treatment significantly improved gentamicin-induced body weight loss in mice and significantly increased urine osmolality compared with mice treated with gentamicin alone. (C and D) SCr and BUN were significantly improved in Nec- 1–treated mice compared with mice treated with gentamicin alone. (E) H&E staining of mouse kidney revealed that Nec-1 treatment significantly attenuated gentamicin-induced tubular injury with improved tubular dilation and reduced cast formation in mice. Mice

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The lack of significant apoptosis in gentamicin-treated mouse mouse kidneys (Figure 3E). In contrast to the predominant kidney could not explain the presence of significant inflam- upregulation of necroptotic signaling, we detected no signifi- matory infiltration and emerging interstitial fibrosis in mouse cant change in cleaved caspase 3 levels in kidney lysates by kidneys after 7 days of gentamicin treatment. We next exam- immunoblotting in mice treated with gentamicin for 7 days ined the kidney tubules in these gentamicin-treated mice in (Figure 3C). These data further support our observations by great detail using TEM. Again, ultrastructural examination did immunofluorescence staining and TEM. not reveal any significant apoptotic features in proximal tu- bules or CDs from the kidneys of mice treated with gentamicin Gentamicin Induces Necroptosis in Cultured Renal for 7 days (data not shown). Tubular Cells However, we observed predominant and unique ultra- To examine the direct effect of gentamicin on tubular cell structural changes in PCs of the CD. The CD PCs appeared necroptosis, cultured LLC-PK1 and mIMCD cells were treated round and enlarged with significantly lucent cytosol with gentamicin at various concentrations. Cell viability was (Figure 2A, b) compared with adjacent intercalated cells evaluated by MTT assay after gentamicin treatment. Our data (ICs) or CD PCs in control mice (Figure 2A, a). Many PCs showed that gentamicin induced cell death in a dose- had disrupted apical plasma membrane (Figure 2B, a, arrows), dependent manner in both cell lines (Figure 4A). The necrop- and the detail of the membrane rupture was viewed under totic pathway was examined next and revealed significantly high magnification (Figure 2B, b and c). Mitochondria in increased expression of RIPK3 and MLKL in a dose-dependent these PCs were mildly swollen, with increased cristae junc- manner in gentamicin-treated LLC-PK1 cells (Figure 4, B and tions compared with adjacent ICs (Figure 2C, b) and that of C, respectively). control PCs (Figure 2C, a). These ultrastructural characteristics We next applied Nec-1, an allosteric inhibitor of RIPK1, to implicate that gentamicin causes necroptosis in CD PCs in mice. gentamicin-treated LLC-PK1 cells.17 Nec-1 significantly Infiltration of neutrophils (Figure 2D, a); excess deposition of blocked gentamicin-induced cell death in LLC-PK1 cells extracellular matrix; and activated fibroblasts containing a (Figure 4C). Gentamicin-induced upregulation of MLKL prominent nucleus, rich cytoplasm, and expanded outreaching and RIPK3 was also impeded by Nec-1 (Figure 4, D and F, processes (Figure 2D,b and c, respectively) were frequently ob- respectively). Nec-1 also blocked the gentamicin-induced served by TEM in the interstitium, surrounding the CDs. translocation of MLKL from the cytosol to the plasma mem- brane in LLC-PK1 cells. Moreover, membrane-accumulated Gentamicin Induces Necroptosis of CD PCs via pMLKL was diminished by Nec-1 treatment in mCCDC11 Upregulation of RIPK3-MLKL Pathway cells (Figure 4G). The intensity of the MLKL and pMLKL Necroptosis is a recently discovered form of regulated necrotic signal in the membrane was quantified by ImageJ cell death involved in the injury of multiple organs, including (Figure 4H). the kidney.34 We detected significant upregulation of MLKL in The specific effect of Nec-1 alleviating gentamicin-induced CDs in gentamicin-treated mice by immunofluorescence necroptosis was further validated by using another optimized staining. Interestingly, the MLKL signals were translocated necroptosis inhibitor, Nec-1s, which has a robust improve- from cytosol to the apical membrane in CD PCs after genta- ment on the metabolic stability and target specificity of micin treatment (Figure 3A). This membrane translocation of RIPK1 in comparison with Nec-1.35 Nec-1s was indeed able MLKL indicates its activation in CD PCs. Indeed, further im- to alleviate gentamicin-induced cell death in a dose-dependent munofluorescence staining using pMLKL confirmed a dra- manner in LLC-PK1 cells. We used specificinhibitors maticaccumulationofpMLKLintheapicalmembranein ferrostatin-1 (Fer-1) and carbobenzoxy-valyl-alanyl-aspartyl- CD PCs in gentamicin-treated mice. Besides, pRIPK3 was (O-methyl)-fluoromethyl ketone (Z-VAD) to block other cell also significantly increased in CD PCs in gentamicin-treated death pathways including ferroptosis and apoptosis, respectively. mice (Figure 3B). In addition to immunostaining, we detected Although Fer-1 was able to rescue erastin-induced cell death significantly increased protein levels of pMLKL, MLKL, from ferroptosis, it failed to do so in gentamicin-induced cell pRIPK3, and RIPK3 in kidney lysates from gentamicin- death in the assay. Although Z-VAD was able to rescue treated mice by immunoblotting (Figure 3, C and D). Sub- cisplatin-induced cell death (apoptosis) in our cultured cells, sequent quantitative real-time PCR revealed significantly it failed to prevent gentamicin-induced cell death increased expression of Ripk3 and Mlkl in gentamicin-treated (Supplemental Figure 2). These data suggest that gentamicin-

were treated with DMSO (Ctrl), gentamicin with DMSO (Genta), and gentamicin with Nec-1 (Genta1Nec-1). (F) Kidney injury score was improved in Nec-1–treated mice compared with mice treated with gentamicin alone. (G) Immunoblotting revealed that Nec-1 treat- ment prevented the gentamicin-induced increase of NGAL level in kidney tissue and urine. Immunoblotting quantification was per- formed using ImageJ software (NIH) and presented in graph (H). Bar represents mean6SEM. *P,0.05, **P,0.01, ***P,0.001 versus control; #P,0.05, ###P,0.001 versus gentamicin. n$3 replicates per group. Statistics was performed using one-way ANOVA for normal distribution data, and the Kruskal–Wallis test has been used for non-normally distributed data. MW, molecular weight.

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A MLKL AQP2 MLKL/AQP2 Ctrl Genta Genta+Nec-1

B Relative protein expression (fold change over internal control) MW Genta+Nec-1 024 6 (KDa) Ctrl Genta

50 MLKL *** 75 MLKL ***# RIPK3 Ctrl 37 Genta * GAPDH Genta+Nec-1 RIPK3 #

C 1.5 2.5 2.5 * *** 2.0 2.0 # # 1.0 1.5 1.5 1.0 1.0 0.5

Mlkl / -Actin 0.5 0.5 Ripk1 / -Actin RIPK3 / -Actin 0 0 0 Ctrl Genta Genta Ctrl Genta Genta Ctrl Genta Genta +Nec-1 +Nec-1 +Nec-1

Figure 6. Inhibition of necroptosis with Nec-1 blocks gentamicin-induced upregulation of MLKL and RIPK3. (A) Immunofluorescence staining revealed that Nec-1 treatment prevented gentamicin-induced apical membrane translocation of MLKL in CD cells. MLKL is stained in red and AQP2 in green. (B) Nec-1 significantly reduced gentamicin-induced upregulation of MLKL and RIPK3 in mouse kidney by immunoblotting. GAPDH was used as loading control. Immunoblotting quantification was performed using ImageJ software (NIH) illustrated in the right panel. (C) Nec-1 significantly blocked gentamicin-induced increase in mRNA of Mlkl and Ripk3 by quantitative real-time PCR. b-Actin was used as internal control. Bar represents mean6SEM. *P,0.05, ***P,0.001 versus control; #P,0.05 versus gentamicin. n$3 replicates per group. Statistical analysis was performed using one-way ANOVA. Ctrl, control; Genta, gentamicin; MW, molecular weight.

2108 JASN JASN 31: 2097–2115, 2020 JASN signi were red) (stained cells Ly6g-positive and F4/80 that revealed 7. Figure rdi pe ae) n eoiino NadCl yimmuno by Col1 and FN of deposition signi and treatment panel), Nec-1 upper (B) alone. in (Genta) (red gentamicin with treated group ae,rsetvl) Q2sandgeni DPs 4 PCs, CD in green stained AQP2 respectively). panel, etsigni ment 31: 2097 etmcnidcdin Gentamicin-induced fi atybokdgnaii-nue lvto fpNF- of elevation gentamicin-induced blocked cantly – 250 135 C AB 15 2020 2115, E F (KDa) MW α-SMA /β-Actin 37 37 50 50 70 70 Genta+Nec-1 0.5 1.0 1.5 2.0 2.5 TNF-α /β-Actin Genta Ctrl 0 0 1 2 3 4 F4/80/AQP2 tlG GM GM Ctrl Ctrl tlG GM+Nec-1 GM Ctrl MGM GM ** * +Nec-1 +Nec-1 fl ## mainand ammation # Ly6g/AQP2 Colal /β-Actin 0.5 1.0 1.5 2.0 2.5 IL-6 /β-Actin 0 0 2 4 6 8 fi tlG GM GM Ctrl tlG GM GM Ctrl rssaealvae yNc1teteti ie A Immuno (A) mice. in treatment Nec-1 by alleviated are brosis 9 6daiio2peyidl tie le cl a,20 bar, Scale blue. stained ,6-diamidino-2-phenylindole *** GAPDH FN a-SMA Coll T-Smad3 p-Smad3 T-NF-κB p-NF-κB * Genta+Nec-1 Genta Ctrl +Nec-1 +Nec-1 α-SMA/AQP2 # # fl k oecnesann F n o1sandrdi ideadlower and middle in red stained Col1 and (FN staining uorescence ,FN, B, fi p-Smad3 p-NF-KB atydcesdi h Nec-1 the in decreased cantly a-SMA

fi Fn /β-Actin IL-1β /β-Actin atyatnae etmcnidcdurglto of upregulation gentamicin-induced attenuated cantly 0 1 2 3 4 0 1 2 3 4 D Coll FN a SA n o1i os inyb muoltig (D) immunoblotting. by kidney mouse in Col1 and -SMA, tlG GM GM Ctrl tlG GM GM Ctrl 02 (fold changeoverinternalcontrol) *** FN/AQP2 * Relative proteinexpression +Nec-1 +Nec-1 # etmcnCue erpoi nKidney in Necroptosis Causes Gentamicin # # # # * * # # *

TGF-β /β-Actin 0.5 1.0 1.5 2.0 2.5 www.jasn.org – rae ru oprdwt the with compared group treated 0 Collal/AQP2 * 46 Genta+Nec-1 Genta Ctrl tlG GM GM Ctrl *** *** fl m +Nec-1 oecnestaining uorescence AI RESEARCH BASIC .()Nc1treat- Nec-1 (C) m. # a -SMA 2109 BASIC RESEARCH www.jasn.org induced cell death is not likely to be mediated by ferroptosis or around the CD in Nec-1–treated mice compared with mice apoptosis. treated with gentamicin alone (Figure 7B). Meanwhile, immu- noblotting revealed that Nec-1 treatment significantly blocked Inhibition of Necroptotic Protein RIPK1 with Nec-1 gentamicin-induced upregulation of phosphorylated nuclear Attenuates Gentamicin-Induced Kidney Injury factor-kB(NF-kB) in mouse kidney. Gentamicin-induced We further tested the effect of necroptosis inhibition in activation of the TGF-b/Smad3 signaling pathway and depo- gentamicin-treated mice. Nec-1 was given to mice through sition of Col1 and FN in mouse kidney were also significantly IP injection 30 minutes before each gentamicin administra- attenuated by Nec-1 as shown by immunoblotting (Figure 7, C tion for a total of 7 days. Mice treated with gentamicin and and D). Consistently, Nec-1 caused a marked reduction in gene Nec-1 had significantly improved body weight and urine os- expression of key inflammatory cytokines—including TNF-a, molality compared with the gentamicin-treated group (Fig- IL-6,andIL-1b—and expression of fibrogenic genes including ure 5, A and B). Nec-1 prevented the elevation of SCr and BUN a-SMA, Col1,andFN in mouse kidney (Figure 7, E and F). induced by gentamicin in mice (Figure 5, C and D). Nec- 1–treated wild-type (WT) mice exhibited no difference in RIPK3-Deficient Mice Are Resistant to body weight and urine osmolality nor in renal function compared Gentamicin-Induced AKI 2 2 with control WT mice (data not shown). Consistent with the im- We next examined the gentamicin-induced AKI in Ripk3 / 2 2 proved renal function, Nec-1 treatment significantly attenuated mice with necroptosis deficiency (Figure 8A). Ripk3 / gentamicin-induced tubular damage in mouse kidneys, as revealed mice show no significant difference compared with WT mice by H&E staining. The kidney injury scores were significantly im- under basal conditions. Increased urine output and reduced urine proved with Nec-1 treatment compared with the gentamicin- osmolality induced by gentamicin as seen in the WT mice were 2 2 treated group (Figure 5, E and F). Furthermore, Nec-1 treatment significantly improved in Ripk3 / mice (Figure 8B). Serum levels 2 2 prevented the elevation of NGAL in gentamicin-treated mice as of creatinine and BUN were significantly reduced in Ripk3 / shown by immunoblotting (Figure 5, G and H). mice treated with gentamicin compared with those of WT We next examined the necroptotic pathway in Nec- (Figure 8, C and D). The level of NGAL in urine and tissue 2 2 1–treated mice. Immunofluorescence staining revealed that was significantly decreased in Ripk3 / mice compared with Nec-1 significantly blocked gentamicin-induced increased WT mice after gentamicin treatment (Figure 8H). 2 2 cellular expression and membrane translocation of MLKL. Gentamicin-treated Ripk3 / mouse kidneys had fewer di- MLKL remained in the cytosol despite gentamicin treatment lated tubules and less cast formation compared with in the Nec-1–treated group (Figure 6A). In addition, immu- gentamicin-treated WT mice. Kidney injury score was signif- 2 2 noblotting revealed that Nec-1 treatment significantly im- icantly decreased in Ripk3 / compared with WTmice treated peded gentamicin-induced elevation of RIPK3 and MLKL in with gentamicin (Figure 8, E and F, upper panel). mouse kidneys (Figure 6B). Quantitative real-time PCR fur- Masson trichrome staining revealed increased collagen in the ther showed that Nec-1 treatment significantly blocked interstitial area in the gentamicin-treated mouse kidneys. The col- 2 2 gentamicin-induced increase in Ripk3 and Mlkl gene expres- lagen deposition was largely impeded in Ripk3 / mice in com- sion in mouse kidneys (Figure 6C). No significant change parison with WT treated with gentamicin (Figure 8E, lower panel). in the expression of ferroptosis key regulators was detected We further examined the necroptotic pathway in WT and 2 2 in the gentamicin-treated group in comparison with control Ripk3 / mice with and without gentamicin treatment. Mem- WT animals (Supplemental Figure 2, E and F). brane accumulation of pMLKL induced by gentamicin was 2 2 largely diminished in Ripk3 / mice, as shown by immuno- Nec-1 Attenuates Gentamicin-Induced Inflammation fluorescence staining (Figure 8G). Immunoblotting revealed and Renal Fibrosis in Mice that RIPK3, MLKL, and pMLKL expression levels were signif- 2 2 Concomitant with reduced necroptosis in the CD, a marked icantly decreased in gentamicin-treated Ripk3 / mice com- reduction of macrophage and neutrophil infiltration around pared with WT mice. Furthermore, the gentamicin-induced the CD was detected by immunofluorescence staining after upregulation of phosphorylated NF-kB was significantly di- 2 2 Nec-1 treatment of mice with gentamicin-induced AKI minished in Ripk3 / mouse kidneys. Significantly increased (Figure 7A and Supplemental Figure 3). Immunostaining of expression level of Col1 and FN was partially blocked in 2 2 a-SMA, FN, and Col1 were also decreased in the interstitium Ripk3 / mice after gentamicin treatment. (Figure 8H). Taken

Statistical analysis was performed using ImageJ. (E) Quantitative real-time PCR revealed that increased expression of proinflammatory cytokines including TNF-a, IL-6,andIL-1b induced by gentamicin was significantly suppressed by Nec-1 treatment in mice. (F) Nec-1 treatment also significantly blocked the elevation of mRNA levels of Fn, Col1,anda-SMA induced by gentamicin in mice by quan- titative real-time PCR. b-Actin was used as internal control. Bar represents mean6SEM. *P,0.05, ***P,0.001 versus control; #P,0.05 versus gentamicin. n$3 replicates per group. Statistical analysis was performed using one-way ANOVA. Ctrl, control; GM, gentamicin; MW, molecular weight.

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ABGentamicin 80mg/kg/mice/day (I.P) 3 ## *** 4000 WT # WT+Genta 3000 *** Ripk3-/- Day 7 2 ### -/- WT ### *** Ripk3 +Genta WT+Genta 2000 -/- 1 Ripk3 (mOsM) 1000 Ripk3-/-+Genta Urine osmolality Urine output (ml) 0 0 D0 D7 D0 D7 C DF 0.5 40 ** 4 *** *** 0.4 30 # 3 0.3 ## ## 20 2 0.2 10 1 0.1 (BUN) (mg/dl) Serum Urea nitrogen 0 0 Kidney injury score 0

Serum creatinine (mg/dl) -/- -/- -/- WT WT WT

Ripk3 -/- +Genta Ripk3 -/- +Genta Ripk3 -/- +Genta WT+Genta WT+Genta WT+Genta Ripk3 Ripk3 Ripk3

E WT Genta Ripk3 -/- Ripk3 -/-+ Genta H&E staining staining Masson’s trichrome

GHpMLKL AQP2 V-ATPase Merge

-/- MW Ripk3 -/- (KDa) WT Genta Ripk3 +Genta WT 75 RIPK3

50 MLKL

50 pMLKL 25 Genta Tissue NGAL

250 FN 135 Coll -/- 70 pNF-κB

Ripk3 70 T-NF-κB 37 GAPDH

25 + Genta Urine NGAL -/- Ripk3

2 2 2 2 Figure 8. Ripk3 / mice are resistant to gentamicin-induced kidney dysfunction and inflammation and renal fibrosis. (A) Ripk3 / mice were treated with or without gentamicin (Genta) as described previously. (B) Metabolic cage experiments revealed that increased urine 2 2 output and decreased urine osmolarity induced by gentamicin treatment for 7 days were alleviated in Ripk3 / mice. (C and D) In- 2 2 creased SCr and serum urea nitrogen induced by gentamicin was attenuated in gentamicin-treated Ripk3 / mice. (E, upper panel, and 2 2 F) Representative images of H&E staining show that Ripk3 / mice have less tubular dilation and cast formation, compared with WT

JASN 31: 2097–2115, 2020 Gentamicin Causes Necroptosis in Kidney 2111 BASIC RESEARCH www.jasn.org together, our results show that inhibiting necroptosis signifi- and chemokines to sustain and/or amplify the inflammatory cantly alleviates renal inflammation, fibrosis, and kidney dys- response.42 Asignificant inflammatory response has been re- function induced by gentamicin in mice. ported in gentamicin-induced nephrotoxicity in experimental animals and in patients.6,45,46 Similarly, in our gentamicin- treated mouse kidneys, we observed significant inflammatory DISCUSSION cell infiltration and dramatically increased expression of in- flammatory cytokines, as well as activation of pNF-kB, which Gentamicin-induced AKI is a devastating complication fre- is a key regulator of inflammation.47 The presence of predom- quently encountered in clinical situations.26,36,37 Despite be- inant necroptosis in CDs is capable of provoking massive kid- ing studied for several decades, there is no effective therapy ney inflammation and fibrosis in the gentamicin-treated available to prevent or treat gentamicin-induced AKI.38,39 It is mouse kidney. well recognized that the main cell death mechanism induced Gentamicin is believed to be freely filtered across the glo- by gentamicin is tubular cell apoptosis, although tubular ne- merulus and then excreted in the urine. Only 5%–10% of crosis is occasionally reported. After entering cells, gentamicin the filtered gentamicin is taken up and sequestered by the accumulates in intracellular organelles including the Golgi proximal tubule cells, where the aminoglycoside can achieve and the endoplasmic reticulum.33 When the concentration concentrations vastly exceeding the concurrent serum con- of gentamicin inside the organelles exceeds a threshold, gen- centration. For many years, the proximal tubule has been tamicin is released into the cytosol. Gentamicin was shown to considered the primary site of gentamicin-induced AKI and act on mitochondria and to promote the formation of reactive proximal tubular damage, mainly apoptosis, was frequently oxygen species, which induce the opening of the mitochon- observed.3,33,48 Interestingly, besides the observed predomi- drial permeability transition pore and trigger the intrinsic nant accumulation of gentamicin in proximal tubule cells, a pathway of apoptosis. Gentamicin can also directly stimulate significant intracellular accumulation of gentamicin in the the production of mitochondrial reactive oxygen species, in- CDs was previously reported.49 How CDs accumulate genta- hibit the respiratory chain and ATP production, and stimulate micin remains to be elucidated. release of cytochrome C and other proapoptotic factors, lead- The kidney CD has been known for decades to be the key ing to apoptosis.40 However, in mice treated with gentamicin segment for water and salt transport and for regulating fluid for 7 days, we were only able to detect occasional apoptosis in balance. Its involvement in kidney injury has not been appre- the proximal tubules. It is well known that a low level of ap- ciated until recently.30,50 Emerging studies have suggested that optosis occurs frequently in the living body, with an estimated renal CD epithelial cells serve as a major source of inflamma- 2003109 apoptotic cells per day, without causing significant tory cytokines during AKI such as ischemia-reperfusion in- inflammation in organs.41,42 Such a low level of tubular cell jury.51,52 We and others have previously shown that disruption apoptosis cannot alone explain the profound inflammation, of the CD PC is “inflammatory” and results in significant fibrosis, and renal injury that occurs in gentamicin-treated damage to the overall kidney structure and function.30,53,54 mice. Therefore, additional and/or alternative injury mecha- Most recently, we reported that integrin-linked kinase (ILK) nism(s) is/are likely involved in gentamicin-induced tubular deficiency in the CD promotes necroptosis. CD necroptosis injury. In this study, we uncover the presence of necroptosis, a resulted in massive inflammation and fibrosis of the kidney in programmed necrosis in the CD in gentamicin-treated mice, ILK knockout mice.55 This study highlights again the impor- adding an important cell death mechanism beside apoptosis tance of the CD integrity to the well-being of the kidney. CD induced by gentamicin. dysfunction, such as ILK deficiency, is sufficient to induce Necroptosis is known to be highly inflammatory.43 During prominent renal failure. Our study reveals that significant nec- necroptosis, the disruption of cell membrane releases a num- roptosis occurred in CD 7 days after gentamicin treatment. ber of cellular contents serving as damage-associated molec- This finding implies that CD injury, which has been over- ular patterns, including mitochondrial DNA and heat-shock looked in the past, may in fact be an important event that proteins.44 These damage-associated molecular patterns trig- mediates massive interstitial inflammation and fibrogenesis ger the production and release of proinflammatory cytokines in gentamicin-induced AKI. under gentamicin treatment. Kidney injury score was given in a blinded fashion as described before. Scale bar, 20 mm. (E, lower panel) 2 2 Masson trichrome staining revealed that collagen fiber deposition in Ripk3 / mice was largely ablated compared with WT under 2 2 gentamicin treatment. (G) Increased pMLKL signal in CD PCs induced by gentamicin largely diminished in Ripk3 / mice under 2 2 gentamicin treatment. Ripk3 / mice appear normal compared with WT mice without gentamicin treatment. (H) Immunoblotting 2 2 showed that, in Ripk3 / mice, the upregulation of necroptosis and inflammation and renal fibrosis pathways induced by gentamicin 2 2 are prevented. For these signal pathways, there was no significant difference between WT and Ripk3 / mice under basal conditions. Bar represents mean6SEM. *P,0.05, **P,0.01; ***P,0.001 versus WT; #P,0.05, ##P,0.01, ###P,0.001 versus gentamicin-treated mice. n$3 replicates per group. Statistics was performed using one-way ANOVA for normal distribution data, and Kruskal–Wallis test has been used for non-normally distributed data. D0, day 0; MW, molecular weight.

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Our data demonstrates the critical involvement of CD nec- Dr. Heyu Ji, and Dr. William W. Jin performed experiments; roptosis in gentamicin-induced AKI. Manipulating the level of Dr. Huihui Huang analyzed data and prepared figures; Dr. Huihui expression, activity, or trafficking of an individual component of Huang, Dr. Ming Huang, William W. Jin, Dr. Jenny Lu, Dr. Teodor the “necrosome,” including RIPK1, RIPK3, and MLKL, have all G. Paunescu,˘ Dr. Yin Xia, and Dr. Junying Yuan edited and revised been shown to affect necroptosis in cells or animals.20,23,24 Be- manuscript; and Dr. Huihui Huang and Dr. Jenny Lu conceived and sides necroptosis, another caspase-independent, regulated ne- designed research, drafted the manuscript, and approved the final crosis, ferroptosis is also known to play an important role in version of the manuscript. mediating tissue injury.56,57 Ferroptosis is characterized by in- creased lipid peroxidation resulting from lack of activity of the glutathione peroxidase 4 that requires glutathione to func- SUPPLEMENTAL MATERIAL tion.58,59 We examined the expression level of two key ferropto- sis regulators GPX4 and SLC7A11 using quantitative PCR, which This article contains the following supplemental material online at did not show any significant changes before and after gentamicin http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019020204/-/ treatment in mouse kidney. We further tested the effect of a DCSupplemental. ferroptosis inhibitor, Fer-1, in our gentamicin-induced cytotox- Supplemental Table 1. List of primers used for quantitative real icity assay. Fer-1 did not show any protective effects against time PCR. gentamicin-induced cell death in vitro. Although our preliminary Supplemental Figure 1. Gentamicin causes kidney tubular injury fl data do not support a major role of ferroptosis in gentamicin- and in ammation with mild proteinuria. induced cell injury, we cannot exclude confidently the involve- Supplemental Figure 2. Gentamicin induces necroptosis other ment of ferroptosis in gentamicin-induced AKI in vivo at this than ferroptosis in cultured renal tubular cells and kidney. point. It would be important and necessary to examine system- Supplemental Figure 3. Nec-1 alleviated gentamicin-induced in- fl fi ically the involvement of ferroptosis as well as other programmed ammatory cell in ltration. cell death pathways in gentamicin-induced tubular injury.

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