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

Significance Statement

Gentamicin-induced AKI is a commonly recognized clinical problem, but the mechanism is not well understood. A mouse model of gentamicin-induced AKI revealed a previously unrecognized role of necroptosis in mediating collecting duct epithelial cell death, interstitial inflammation, and fibrosis. Importantly, either inhibiting a necroptotic pathway activator RIPK1 kinase with its inhibitor Nec-1 or deleting a key necroptotic gene, Ripk3, significantly attenuated gentamicin-induced AKI in mice and in cultured porcine and murine kidney tubular cells. Identification of a novel programmed necroptosis pathway in gentamicin-induced renal tubule injury could provide a new therapeutic target.

Visual Abstract

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 apoptosis 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 cell death 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 necroptosis, 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 addition, 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 inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, 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.

Aminoglycosides, such as gentamicin, are one of the most commonly used classes of antibiotics to treat gram-negative bacterial infection. However, its clinical application has been greatly limited by its associated nephrotoxicity. The incidence of gentamicin-induced AKI ranges from 2% to as high as 55%.¹ It is estimated that up to 30% of patients treated with gentamicin for more than 7 days exhibit signs of renal impairment.² Multiple efforts to prevent gentamicin-induced AKI have not been effective due to the lack of understanding of the specific mechanism underlying gentamicin-induced tubular injury. It was observed a long time ago that gentamicin was primarily taken up by and accumulated in proximal tubular cells and caused the proximal tubular injury. For many years, it was believed that the main mechanism of gentamicin-induced AKI is tubular cell apoptosis, which occurs predominantly in proximal tubules in experimental animals and patients treated with gentamicin.^3,4 However, treatment to inhibit apoptosis using various methods only partially improves gentamicin-induced AKI in animals.^5–7 Additional and/or alternative tubular injury mechanisms underlying gentamicin-induced AKI are under active investigation.⁸

Very interestingly, unlike other types of acute tubular injury resulting in oliguric AKI, gentamicin-induced AKI is frequently associated with polyuria and reduced urine osmolality, which implicates a possible defect of the urinary concentration mechanism, a main function of the collecting duct (CD). Whether gentamicin-induced AKI could involve other type(s) of tubular cell injury mechanisms and/or other tubular segments, such as CDs, besides proximal tubules is not clear. Using an anti-gentamicin antibody to stain gentamicin-treated mouse kidney, it was found that, besides a predominant accumulation of gentamicin in proximal tubular cells, there was a significant accumulation of gentamicin in the CDs. Those CD cells that accumulated gentamicin appeared “round” or swollen with prominent nuclei.⁹ However, how the CD tubular cells took up gentamicin and whether they contributed to gentamicin-induced AKI remains unknown. This study suggests that uptake of gentamicin by CD leads to CD necroptosis. CD necroptosis likely contributes critically to the development of massive inflammation and interstitial fibrosis in gentamicin-induced AKI.

Several types of cell death, including apoptosis and necrosis, occur during kidney tubular injury. Apoptosis is a regulated cell death that has been well studied for decades in many tissues, including the kidney.^10,11 Tubular cell apoptosis was once considered to be a key form of cell death leading to CKD. However, this point of view was recently challenged given the discovery of a regulated necrosis pathway, necroptosis. Necroptosis is the most characterized pathway of regulated necrosis in higher eukaryotic cells.^12,13 It is implicated in a wide range of high-grade tissue injuries such as myocardial ischemia and reperfusion injury, amyotrophic lateral sclerosis, sepsis, and intestinal inflammation.^14–16 The best-studied necroptotic pathway is the TNF-induced necroptosis through activation of TNF receptor 1.^17,18 Induction of necroptosis is mediated through activation of TNF receptor 1 and subsequent activation and interaction of the receptor-interacting protein kinase 1 (RIPK1) and RIPK3. RIPK1 and RIPK3 interaction leads to recruitment and phosphorylation of the mixed lineage kinase domain-like pseudokinase (MLKL), forming the necrosome. Phosphorylated MLKL (pMLKL) becomes oligomerized and then translocates from the cytosol to the plasma membrane where it disrupts the latter.¹⁹ In addition, a very recent study has demonstrated the existence of a repair mechanism mediated by the endosomal sorting complex required for transport (ESCRT-III). Through shedding of MLKL-damaged membrane, ESCRT-III is able to sustain cell survival despite MLKL activation.²⁰ When cells undergo necroptosis, they present with organelle and cell swelling, permeabilization of the plasma membrane, and spilling of intracellular contents.^21,22 Emerging studies have revealed that blocking necroptosis using a RIPK1 inhibitor or RIPK3-deficient mice largely alleviated multiple kidney injuries induced by ischemia reperfusion; chemical injury from cisplatin, cyclosporine, and contrast dye; and by unilateral ureteral obstruction.^23–25

It has been well appreciated that gentamicin causes significant AKI and some of the patients with gentamicin-induced AKI progress to ESKD. Massive inflammation and tubular damage were reported in animals and patients treated with gentamicin.²⁶ Whether necroptosis, in addition to apoptosis, is involved in gentamicin-induced tubular injury has not been elucidated. To understand the pathologic mechanism underlying gentamicin-induced AKI, we performed a study to re-examine the tubular cell injury in gentamicin-treated mice. We demonstrated the presence of widespread interstitial inflammation, fibrosis, and significant renal dysfunction after 7 days of treatment with gentamicin in mice. Besides a low grade of apoptosis occurring in proximal tubules, we have unexpectedly detected widespread necroptosis in principal cells (PCs) of the CDs in gentamicin-treated animals. We further show that gentamicin induced upregulation of necroptotic signals, and inhibiting necroptosis by RIPK1 inhibitor necrostatin-1 (Nec-1) or RIPK3 deficiency significantly alleviated gentamicin-induced inflammation, interstitial fibrosis, and kidney dysfunction.

Methods

Animals

C57/BL6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were given free access to food and water under a 12-hour light/dark cycle. Ripk3^−/− mice were kindly provided by Dr. Vishva Dixit from Genentech.²⁷ All animal experiments were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Massachusetts General Hospital (MGH) Subcommittee on Research Animal Care. All mice were anesthetized using isoflurane. Mice subjected to perfusion were perfused through the left cardiac ventricle with PBS containing 0.9% sodium chloride in 10 mM phosphate buffer at pH 7.4, at a rate of 13–17 ml/min for 4 minutes, followed by modified paraformaldehyde-lysine-periodate (PLP) for 4 minutes. Modified PLP consisted of 4% paraformaldehyde, 75 mM lysine-hydrochloride, 10 mM sodium periodate, and 0.15 M sucrose in 37.5 mM sodium phosphate. Kidneys from perfusion-fixed mice and mice that were not perfused were fixed with modified PLP at 4°C overnight, washed with PBS for 3×3 hours, and stored in PBS containing 0.02% sodium azide until use.

Experimental Groups and Drug Treatments

C57/BL6J mice (8 weeks old) were injected intraperitoneally (IP) with gentamicin (Thermo Fisher Scientific, Waltham, MA) at 80 mg/kg per day for 7 days to induce kidney injury. For the Nec-1 (Sigma-Aldrich, St. Louis, MO) experiment, mice were divided into three groups. The first group was the control and was given vehicle (DMSO, 1.65 mg/kg per day) through IP injection for 7 days. The second group was pretreated with vehicle (DMSO, 1.65 mg/kg per day) 30 minutes before IP gentamicin injection at 80 mg/kg per day for 7 days. The third group was pretreated with Nec-1 (1.65 mg/kg per day) 30 minutes before IP gentamicin injection at 80 mg/kg per day for 7 days.

Metabolic Cage Studies

C57/BL6J mice (8 weeks old) in the control and gentamicin treatment groups were subjected to 24-hour metabolic cage monitoring. Urine samples were collected and water and food intake was recorded. Urine osmolality was measured with a Vapor Pressure Osmometer 5520 (Wescor, Logan, UT).

Urinary Analysis

Urinary analysis was performed as previously described.²⁸ Spot urine samples (2 µl) from each group were mixed with 5 µl of SDS sample loading buffer, and subjected to 10% SDS-PAGE. Gels were stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA) for 1 hour and washed with double-distilled water for 1 hour. BSA (Santa Cruz Biotechnology, Dallas, TX) was used for the albumin control band.

Serum Creatinine and BUN Measurements

Terminal blood collection was performed. Serum was separated using a BD Microtainer (Becton-Dickinson, Franklin Lakes, NJ) and stored at −80°C. Serum creatinine (SCr) levels were measured with the QuantiChrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s protocol. BUN levels were measured with the Stanbio Urea Nitrogen Kit (Stanbio Laboratory, Boerne, TX) according to the manufacturer’s protocol.

Hematoxylin and Eosin Staining

Nonperfused, modified PLP-fixed kidney tissues were embedded in paraffin blocks and sectioned at 5-µm thickness. Sections were used for hematoxylin and eosin (H&E) staining according to the manufacturer’s protocol. Briefly, sections were deparaffinized with xylene, rehydrated in serial dilutions of ethanol, and stained with the Weigert iron hematoxylin set (Sigma-Aldrich) for staining nuclei. Sections were rinsed with normal tap water, differentiated with 0.3% acid alcohol, and then “blued” with Scott’s tap water (238 mM sodium bicarbonate and 29 mM magnesium sulfate in distilled water) for 2 minutes. Sections were then treated with 1% eosin (Sigma-Aldrich) for cytosol staining, dehydrated, and mounted.

Picrosirius Red Staining and Masson Trichrome Staining

Staining was performed with the Picrosirius Red Stain Kit (Polysciences Inc, Warrington, PA) or Trichrome Stain Kit (Masson, Sigma-Aldrich) according to the manufacturer’s protocol. The sections were examined with a Zeiss LSM800 confocal microscope (Carl Zeiss Microscopy, Thornwood, NY) under normal and polarized light. Analysis of staining intensity was performed using ImageJ software (NIH, Bethesda, MD).

Immunofluorescence Staining and Immunoblotting

Perfused, modified PLP-fixed kidney tissues were embedded in ornithine carbamyl transferase or paraffin and sectioned at 5-µm thickness for immunofluorescence staining. Cryosections were used to detect MLKL using an anti-MLKL antibody diluted 1:500 (#AP14272b; Abgent, San Diego, CA). The primary antibodies used for paraffin sections were anti-AQP2 diluted 1:1600 (#sc-9882; Santa Cruz Biotechnology), anti V-ATPase (provided by Dr. Dennis Brown) diluted at 1:800, anti-fibronectin (anti-FN) diluted 1:2000 (#F3648; Sigma-Aldrich), anti–α-smooth muscle actin (anti–α-SMA) diluted 1:2000 (#A5228; Sigma-Aldrich), anti–collagen type I (anti-Col1) diluted 1:200 (#C2456; Sigma-Aldrich), anti-F4/80 diluted 1:100 (#14-4801-85; Invitrogen), anti-MLKL (phosphor S358) diluted 1:1500 (#ab196436; Abcam, Cambridge, United Kingdom), anti–phosphorylated RIPK3 (anti-pRIPK3; Thr231/Ser232) diluted 1:500 (#57220; Cell Signaling Technology, Danvers, MA). Briefly, for cryosections, the slides were rehydrated in PBS, incubated with 1% SDS for 4 minutes, and washed with PBS. For paraffin sections, the slides were deparaffinized and rehydrated as described above. Antigen retrieval was performed by incubating slides in Tris-EDTA buffer (10 mM 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 washed with PBS. The slides from both cryosections and paraffin sections were blocked with 1% (w/v) BSA in PBS and then incubated with primary antibodies at 4°C overnight. After washing with PBS, slides were incubated with fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 1 hour. After washing with PBS, the slides were mounted with 4′,6-diamidino-2-phenylindole. Fluorescence images were acquired using a Zeiss LSM800 confocal microscope.

Immunoblotting was performed as previously described using the following antibodies²⁹: anti-FN diluted 1:1000 (#F3648), anti–phosphorylated-Smad3 (S423/425) diluted 1:1000 (#9520; Abcam), anti-Smad3 diluted 1:1000 (#9523; Cell Signaling Technology), anti–α–SMA diluted 1:1000 (#A5228), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) diluted 1:20,000 (#2118; Cell Signaling Technology), anti–β-actin diluted 1:10,000 (#4967; Cell Signaling Technology), anti-MLKL (phosphor S358) diluted 1:1500 (#ab196436), anti-MLKL diluted 1:1000 (#MABC604; Millipore-Sigma, Burlington, MA), anti–neutrophil gelatinase-associated lipocalin (anti-NGAL) diluted 1:1000 (#AF-1857; R&D Systems, Minneapolis, MN), anti–phospho-NF-κB diluted 1:1000 (#3033; Cell Signaling Technology), anti–T-NF-κB diluted 1:1000 (#8242; Cell Signaling Technology), anti–caspase 3 diluted 1:1000 (#9662; Cell Signaling Technology), anti-RIPK3 diluted 1:1000 (#sc-374639; Santa Cruz Biotechnology), and anti-pRIPK3 (Thr231/Ser232) diluted 1:1000 (#57220).

RNA Extraction and Quantitative Real-Time PCR

RNA was extracted from control and treated mice. Briefly, mouse kidneys from each group were homogenized in TRIzol (Invitrogen) and separated using chloroform after centrifugation. RNA was precipitated with isopropanol, washed with 70% ethanol, and dissolved in diethyl pyrocarbonate–treated water. cDNA synthesis was carried out with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using the QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, CA). mRNA levels for each gene were normalized to the expression levels of the housekeeping gene β-actin. Relative mRNA expression was determined using the Ct method. The list of primer sequences is shown in Supplemental Table 1.

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was performed as previously described.³⁰ Briefly, kidney slices were fixed as described above, rinsed in PBS, and postfixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer for 24 hours at 4°C. The slices were rinsed with 0.1 M sodium cacodylate buffer and infiltrated with 1% osmium tetroxide in cacodylate buffer for 1 hour at room temperature. Tissue blocks were rinsed again in cacodylate buffer, fully dehydrated through a series of graded ethanols, dehydrated briefly with 100% propylene oxide, and preinfiltrated with a 1:1 mix of Eponate resin (Ted Pella, Redding, CA) and propylene oxide overnight on a gentle rotator. The following day, specimens were infiltrated with fresh Eponate resin, embedded in flat molds with fresh Eponate, and allowed to polymerize for 24–48 hours at 60°C. Thin sections were cut using an EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany), collected onto formvar-coated grids, stained with uranyl acetate and lead citrate, and examined under a JEM 1011 transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV. Images were collected using an AMT digital imaging system (Advanced Microscopy Techniques, Danvers, MA).

Cell Culture and Cell Viability Assay

LLC-PK1, mIMCD, and mCCDC11 cells were cultured in DMEM medium (Thermo Fisher Scientific) containing 10% FBS, in a 5% carbon dioxide humidified atmosphere at 37°C. Cells were treated with gentamicin at different doses for 48 hours. Cells were harvested, lysed in lysis buffer, and subjected to immunoblotting. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.³¹ Briefly, cultured cells were seeded onto 96-well plates and grown to approximately 80% confluence. The cells were then treated with different doses of gentamicin, gentamicin with vehicle, or Nec-1. At 48 hours after treatment, cells were incubated with 20 μl 5 mg/ml MTT (Affymetrix, Santa Clara, CA) for 3 hours. After incubation, cells were treated with the MTT solvent DMSO for 5 minutes at room temperature. Absorbance was measured as OD at 570 nm on a SpectraMax Multi-Mode microplate reader (Molecular Devices, San Jose, CA).

Statistical Analyses

Data were analyzed using an unpaired design. For normal distribution, we used t test for two groups and one-way ANOVA for more than two groups. For non-normal distribution, we used a nonparametric test. To compare two groups, we used the Mann–Whitney test, and to compare three groups, the Kruskal–Wallis test. Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software, San Diego, CA). A P value <0.05 was considered statistically significant. Individual P values are specified in the figure legends.

Results

Gentamicin Causes Kidney Dysfunction, Inflammation, and Fibrosis

C57/B6 mice were injected with 80 mg/kg gentamicin per day IP for 7 days. Metabolic parameters were obtained using metabolic cages. The urine output was significantly increased to twice the control value, and urine osmolality was reduced to half, indicating impaired urine concentration in gentamicin-treated mice (Figure 1A). Renal function was assessed by measuring the level of SCr and BUN. SCr was increased from 0.2 mg/dl in the control to 0.6 mg/dl in the gentamicin-treated mice (Figure 1B, left panel). BUN was significantly elevated in gentamicin-treated mice compared with the control (Figure 1B, right panel). The expression level of NGAL in both kidney and urine was significantly elevated in gentamicin-treated mice (Figure 1C). Moreover, mild proteinuria was detected in gentamicin-treated mice by Coomassie blue staining of SDS-PAGE from spot urine (Supplemental Figure 1C).

Figure 1.

Gentamicin causes kidney dysfunction with inflammation and renal fibrosis. Gentamicin (80 mg/kg per day) was given to C57BL/6 mice (n=6) 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 gentamicin-treated mice. Brush border disruption, tubular dilation, tubular cell injury, and cast formation were observed in gentamicin-treated mice. Scale bar, 20 μm. (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 μm. Immunofluorescence staining of gentamicin-treated mouse kidney with antibodies against FN, α-SMA, and Col1 (right panels). All sections were costained with anti-AQP2 antibody (green) to identify CDs in the renal cortex. Blue represents 4′,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 μm. (H) Increased mRNA level of a-SMA, FN, and Col1 was detected in gentamicin-treated mice by quantitative real-time PCR. The mRNA level of proinflammatory cytokines including TNF-a, IL-6, and IL-1β was significantly increased by quantitative real-time PCR analysis in the gentamicin-treated group compared with the control group. Bar values represent mean±SEM, n=6 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.

H&E staining revealed the presence of dilated tubules and tubular atrophy in gentamicin-treated kidneys. Occasionally, cellular debris was found in dilated cortical tubules (Figure 1D). Lower magnification of these images is shown in the Supplemental Figure 1A. The kidney injury score was evaluated independently by three researchers and the score was significantly increased in gentamicin-treated mice (Figure 1E).

We next examined the interstitial fibrosis in the gentamicin-treated mouse kidney. Picrosirius red staining revealed dramatically increased deposition of collagen fibrils in gentamicin-treated mouse kidney (Figure 1F, left panel). Immunofluorescence staining with antibodies against extracellular matrix including collagen type I (Col1) and fibronectin (FN), and the myofibroblast marker alpha-smooth muscle actin (α-SMA) showed that the expression of those proteins was significantly increased in the interstitium around CDs after gentamicin treatment (Figure 1F, right panel).

It has been reported that maladaptive inflammatory response promotes interstitial fibrosis in many AKI models, including animals and patients treated with gentamicin .³² Immunofluorescence staining using antibodies against the macrophage marker F4/80 and the leukocyte/neutrophil marker Ly6g revealed significant infiltration of macrophages and neutrophils in gentamicin-treated mouse kidney (Figure 1G). Inflammatory cells were quantified by ImageJ (Supplemental Figure 1B). Quantitative real-time PCR revealed significantly increased expression of fibrogenic genes a-SMA, FN, and Col1, as well as of several proinflammatory markers (TNF-α, IL-6, and IL-1β), in gentamicin-treated mouse kidneys (Figure 1H). These results demonstrate that gentamicin induces significant renal inflammation and interstitial fibrosis in mice.

Tubular Cell Injury Revealed by TEM in Gentamicin-Treated Mouse Kidney

Gentamicin was reported to cause apoptosis of proximal tubular cells.³³ However, when we examined the apoptosis of tubular cells in mice treated with gentamicin for 7 days by immunofluorescence staining, we only detected very few apoptotic cells that were stained for cleaved caspase 3, and apoptotic cells were barely detectable in the CD (data not shown). The lack of significant apoptosis in gentamicin-treated mouse kidney could not explain the presence of significant inflammatory infiltration and emerging interstitial fibrosis in mouse kidneys after 7 days of gentamicin treatment. We next examined the kidney tubules in these gentamicin-treated mice in great detail using TEM. Again, ultrastructural examination did not reveal any significant apoptotic features in proximal tubules or CDs from the kidneys of mice treated with gentamicin for 7 days (data not shown).

However, we observed predominant and unique ultrastructural changes in PCs of the CD. The CD PCs appeared round and enlarged with significantly lucent cytosol (Figure 2A, b) compared with adjacent intercalated cells (ICs) or CD PCs in control mice (Figure 2A, a). Many PCs had disrupted apical plasma membrane (Figure 2B, a, arrows), and the detail of the membrane rupture was viewed under high magnification (Figure 2B, b and c). Mitochondria in these PCs were mildly swollen, with increased cristae junctions compared with adjacent ICs (Figure 2C, b) and that of control PCs (Figure 2C, a). These ultrastructural characteristics implicate that gentamicin causes necroptosis in CD PCs in mice. Infiltration of neutrophils (Figure 2D, a); excess deposition of extracellular matrix; and activated fibroblasts containing a prominent nucleus, rich cytoplasm, and expanded outreaching processes (Figure 2D,b and c, respectively) were frequently observed by TEM in the interstitium, surrounding the CDs.

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.

Gentamicin Induces Necroptosis of CD PCs via Upregulation of RIPK3-MLKL Pathway

Necroptosis is a recently discovered form of regulated necrotic cell death involved in the injury of multiple organs, including the kidney.³⁴ We detected significant upregulation of MLKL in CDs in gentamicin-treated mice by immunofluorescence staining. Interestingly, the MLKL signals were translocated from cytosol to the apical membrane in CD PCs after gentamicin treatment (Figure 3A). This membrane translocation of MLKL indicates its activation in CD PCs. Indeed, further immunofluorescence staining using pMLKL confirmed a dramatic accumulation of pMLKL in the apical membrane in CD PCs in gentamicin-treated mice. Besides, pRIPK3 was also significantly increased in CD PCs in gentamicin-treated mice (Figure 3B). In addition to immunostaining, we detected significantly increased protein levels of pMLKL, MLKL, pRIPK3, and RIPK3 in kidney lysates from gentamicin-treated mice by immunoblotting (Figure 3, C and D). Subsequent quantitative real-time PCR revealed significantly increased expression of Ripk3 and Mlkl in gentamicin-treated mouse kidneys (Figure 3E). In contrast to the predominant upregulation of necroptotic signaling, we detected no significant change in cleaved caspase 3 levels in kidney lysates by immunoblotting in mice treated with gentamicin for 7 days (Figure 3C). These data further support our observations by immunofluorescence staining and TEM.

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 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 mean±SEM, *P<0.05, **P<0.01 versus control. Statistical analysis was performed using t test. MW, molecular weight.

Gentamicin Induces Necroptosis in Cultured Renal Tubular Cells

To examine the direct effect of gentamicin on tubular cell necroptosis, cultured LLC-PK1 and mIMCD cells were treated with gentamicin at various concentrations. Cell viability was evaluated by MTT assay after gentamicin treatment. Our data showed that gentamicin induced cell death in a dose-dependent manner in both cell lines (Figure 4A). The necroptotic pathway was examined next and revealed significantly increased expression of RIPK3 and MLKL in a dose-dependent manner in gentamicin-treated LLC-PK1 cells (Figure 4, B and C, respectively).

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; n=6. (B) Gentamicin induced upregulation of MLKL and RIPK3 in a dose-dependent manner in cultured cells by immunoblotting. Quantification of immunoblots was performed using ImageJ and illustrated in (E). *P<0.05 versus control, **P<0.01versus control, ***P<0.001 versus control, ^&P<0.05 versus 0.2 mM gentamicin, ^&&&P<0.001versus 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 measured by the MTT assay. Gentamicin (GM; 2 mM) was incubated with or without 60 μM 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 gentamicin+Nec-1 (30 μM). (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.

We next applied Nec-1, an allosteric inhibitor of RIPK1, to gentamicin-treated LLC-PK1 cells.¹⁷ Nec-1 significantly blocked gentamicin-induced cell death in LLC-PK1 cells (Figure 4C). Gentamicin-induced upregulation of MLKL and RIPK3 was also impeded by Nec-1 (Figure 4, D and F, respectively). Nec-1 also blocked the gentamicin-induced translocation of MLKL from the cytosol to the plasma membrane in LLC-PK1 cells. Moreover, membrane-accumulated pMLKL was diminished by Nec-1 treatment in mCCDC11 cells (Figure 4G). The intensity of the MLKL and pMLKL signal in the membrane was quantified by ImageJ (Figure 4H).

The specific effect of Nec-1 alleviating gentamicin-induced necroptosis was further validated by using another optimized necroptosis inhibitor, Nec-1s, which has a robust improvement on the metabolic stability and target specificity of RIPK1 in comparison with Nec-1.³⁵ Nec-1s was indeed able to alleviate gentamicin-induced cell death in a dose-dependent manner in LLC-PK1 cells. We used specific inhibitors ferrostatin-1 (Fer-1) and carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethyl ketone (Z-VAD) to block other cell death pathways including ferroptosis and apoptosis, respectively. Although Fer-1 was able to rescue erastin-induced cell death from ferroptosis, it failed to do so in gentamicin-induced cell death in the assay. Although Z-VAD was able to rescue cisplatin-induced cell death (apoptosis) in our cultured cells, it failed to prevent gentamicin-induced cell death (Supplemental Figure 2). These data suggest that gentamicin-induced cell death is not likely to be mediated by ferroptosis or apoptosis.

Inhibition of Necroptotic Protein RIPK1 with Nec-1 Attenuates Gentamicin-Induced Kidney Injury

We further tested the effect of necroptosis inhibition in gentamicin-treated mice. Nec-1 was given to mice through IP injection 30 minutes before each gentamicin administration for a total of 7 days. Mice treated with gentamicin and Nec-1 had significantly improved body weight and urine osmolality compared with the gentamicin-treated group (Figure 5, A and B). Nec-1 prevented the elevation of SCr and BUN induced by gentamicin in mice (Figure 5, C and D). Nec-1–treated wild-type (WT) mice exhibited no difference in body weight and urine osmolality nor in renal function compared with control WT mice (data not shown). Consistent with the improved renal function, Nec-1 treatment significantly attenuated gentamicin-induced tubular damage in mouse kidneys, as revealed by H&E staining. The kidney injury scores were significantly improved with Nec-1 treatment compared with the gentamicin-treated group (Figure 5, E and F). Furthermore, Nec-1 treatment prevented the elevation of NGAL in gentamicin-treated mice as shown by immunoblotting (Figure 5, G and H).

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 were treated with DMSO (Ctrl), gentamicin with DMSO (Genta), and gentamicin with Nec-1 (Genta+Nec-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 treatment prevented the gentamicin-induced increase of NGAL level in kidney tissue and urine. Immunoblotting quantification was performed using ImageJ software (NIH) and presented in graph (H). Bar represents mean±SEM. *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.

We next examined the necroptotic pathway in Nec-1–treated mice. Immunofluorescence staining revealed that Nec-1 significantly blocked gentamicin-induced increased cellular expression and membrane translocation of MLKL. MLKL remained in the cytosol despite gentamicin treatment in the Nec-1–treated group (Figure 6A). In addition, immunoblotting revealed that Nec-1 treatment significantly impeded gentamicin-induced elevation of RIPK3 and MLKL in mouse kidneys (Figure 6B). Quantitative real-time PCR further showed that Nec-1 treatment significantly blocked gentamicin-induced increase in Ripk3 and Mlkl gene expression in mouse kidneys (Figure 6C). No significant change in the expression of ferroptosis key regulators was detected in the gentamicin-treated group in comparison with control WT animals (Supplemental Figure 2, E and F).

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. β-Actin was used as internal control. Bar represents mean±SEM. *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.

Nec-1 Attenuates Gentamicin-Induced Inflammation and Renal Fibrosis in Mice

Concomitant with reduced necroptosis in the CD, a marked reduction of macrophage and neutrophil infiltration around the CD was detected by immunofluorescence staining after Nec-1 treatment of mice with gentamicin-induced AKI (Figure 7A and Supplemental Figure 3). Immunostaining of α-SMA, FN, and Col1 were also decreased in the interstitium around the CD in Nec-1–treated mice compared with mice treated with gentamicin alone (Figure 7B). Meanwhile, immunoblotting revealed that Nec-1 treatment significantly blocked gentamicin-induced upregulation of phosphorylated nuclear factor-κB (NF-κB) in mouse kidney. Gentamicin-induced activation of the TGF-β/Smad3 signaling pathway and deposition of Col1 and FN in mouse kidney were also significantly attenuated by Nec-1 as shown by immunoblotting (Figure 7, C and D). Consistently, Nec-1 caused a marked reduction in gene expression of key inflammatory cytokines—including TNF-α, IL-6, and IL-1β—and expression of fibrogenic genes including a-SMA, Col1, and FN in mouse kidney (Figure 7, E and F).

Figure 7.

Gentamicin-induced inflammation and fibrosis are alleviated by Nec-1 treatment in mice. (A) Immunofluorescence staining revealed that F4/80 and Ly6g-positive cells (stained red) were significantly decreased in the Nec-1–treated group compared with the group treated with gentamicin (Genta) alone. (B) Nec-1 treatment significantly attenuated gentamicin-induced upregulation of α-SMA (red in upper panel), and deposition of FN and Col1 by immunofluorescence staining (FN and Col1 stained red in middle and lower panel, respectively). AQP2 stained green in CD PCs, 4′,6-diamidino-2-phenylindole stained blue. Scale bar, 20 μm. (C) Nec-1 treatment significantly blocked gentamicin-induced elevation of pNF-κB, FN, α-SMA, and Col1 in mouse kidney by immunoblotting. (D) Statistical analysis was performed using ImageJ. (E) Quantitative real-time PCR revealed that increased expression of proinflammatory cytokines including TNF-α, IL-6, and IL-1β 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, and a-SMA induced by gentamicin in mice by quantitative real-time PCR. β-Actin was used as internal control. Bar represents mean±SEM. *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.

RIPK3-Deficient Mice Are Resistant to Gentamicin-Induced AKI

We next examined the gentamicin-induced AKI in Ripk3^−/− mice with necroptosis deficiency (Figure 8A). Ripk3^−/− mice show no significant difference compared with WT mice under basal conditions. Increased urine output and reduced urine osmolality induced by gentamicin as seen in the WT mice were significantly improved in Ripk3^−/− mice (Figure 8B). Serum levels of creatinine and BUN were significantly reduced in Ripk3^−/− mice treated with gentamicin compared with those of WT (Figure 8, C and D). The level of NGAL in urine and tissue was significantly decreased in Ripk3^−/− mice compared with WT mice after gentamicin treatment (Figure 8H).

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 output and decreased urine osmolarity induced by gentamicin treatment for 7 days were alleviated in Ripk3^−/− mice. (C and D) Increased SCr and serum urea nitrogen induced by gentamicin was attenuated in gentamicin-treated Ripk3^−/− mice. (E, upper panel, and F) Representative images of H&E staining show that Ripk3^−/− mice have less tubular dilation and cast formation, compared with WT under gentamicin treatment. Kidney injury score was given in a blinded fashion as described before. Scale bar, 20 μm. (E, lower panel) Masson trichrome staining revealed that collagen fiber deposition in Ripk3^−/− mice was largely ablated compared with WT under gentamicin treatment. (G) Increased pMLKL signal in CD PCs induced by gentamicin largely diminished in Ripk3^−/− mice under gentamicin treatment. Ripk3^−/− mice appear normal compared with WT mice without gentamicin treatment. (H) Immunoblotting showed that, in Ripk3^−/− mice, the upregulation of necroptosis and inflammation and renal fibrosis pathways induced by gentamicin are prevented. For these signal pathways, there was no significant difference between WT and Ripk3^−/− mice under basal conditions. Bar represents mean±SEM. *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.

Gentamicin-treated Ripk3^−/− mouse kidneys had fewer dilated tubules and less cast formation compared with gentamicin-treated WT mice. Kidney injury score was significantly decreased in Ripk3^−/− compared with WT mice treated with gentamicin (Figure 8, E and F, upper panel).

Masson trichrome staining revealed increased collagen in the interstitial area in the gentamicin-treated mouse kidneys. The collagen deposition was largely impeded in Ripk3^−/− mice in comparison with WT treated with gentamicin (Figure 8E, lower panel).

We further examined the necroptotic pathway in WT and Ripk3^−/− mice with and without gentamicin treatment. Membrane accumulation of pMLKL induced by gentamicin was largely diminished in Ripk3^−/− mice, as shown by immunofluorescence staining (Figure 8G). Immunoblotting revealed that RIPK3, MLKL, and pMLKL expression levels were significantly decreased in gentamicin-treated Ripk3^−/− mice compared with WT mice. Furthermore, the gentamicin-induced upregulation of phosphorylated NF-κB was significantly diminished in Ripk3^−/− mouse kidneys. Significantly increased expression level of Col1 and FN was partially blocked in Ripk3^−/− mice after gentamicin treatment. (Figure 8H). Taken together, our results show that inhibiting necroptosis significantly alleviates renal inflammation, fibrosis, and kidney dysfunction induced by gentamicin in mice.

Discussion

Gentamicin-induced AKI is a devastating complication frequently encountered in clinical situations.^26,36,37 Despite being studied for several decades, there is no effective therapy available to prevent or treat gentamicin-induced AKI.^38,39 It is well recognized that the main cell death mechanism induced by gentamicin is tubular cell apoptosis, although tubular necrosis is occasionally reported. After entering cells, gentamicin accumulates in intracellular organelles including the Golgi and the endoplasmic reticulum.³³ When the concentration of gentamicin inside the organelles exceeds a threshold, gentamicin is released into the cytosol. Gentamicin was shown to act on mitochondria and to promote the formation of reactive oxygen species, which induce the opening of the mitochondrial permeability transition pore and trigger the intrinsic pathway of apoptosis. Gentamicin can also directly stimulate the production of mitochondrial reactive oxygen species, inhibit the respiratory chain and ATP production, and stimulate release of cytochrome C and other proapoptotic factors, leading to apoptosis.⁴⁰ However, in mice treated with gentamicin for 7 days, we were only able to detect occasional apoptosis in the proximal tubules. It is well known that a low level of apoptosis occurs frequently in the living body, with an estimated 200×10⁹ apoptotic cells per day, without causing significant inflammation in organs.^41,42 Such a low level of tubular cell apoptosis cannot alone explain the profound inflammation, fibrosis, and renal injury that occurs in gentamicin-treated mice. Therefore, additional and/or alternative injury mechanism(s) is/are likely involved in gentamicin-induced tubular injury. In this study, we uncover the presence of necroptosis, a programmed necrosis in the CD in gentamicin-treated mice, adding an important cell death mechanism beside apoptosis induced by gentamicin.

Necroptosis is known to be highly inflammatory.⁴³ During necroptosis, the disruption of cell membrane releases a number of cellular contents serving as damage-associated molecular patterns, including mitochondrial DNA and heat-shock proteins.⁴⁴ These damage-associated molecular patterns trigger the production and release of proinflammatory cytokines and chemokines to sustain and/or amplify the inflammatory response.⁴² A significant inflammatory response has been reported in gentamicin-induced nephrotoxicity in experimental animals and in patients.^6,45,46 Similarly, in our gentamicin-treated mouse kidneys, we observed significant inflammatory cell infiltration and dramatically increased expression of inflammatory cytokines, as well as activation of pNF-κB, which is a key regulator of inflammation.⁴⁷ The presence of predominant necroptosis in CDs is capable of provoking massive kidney inflammation and fibrosis in the gentamicin-treated mouse kidney.

Gentamicin is believed to be freely filtered across the glomerulus and then excreted in the urine. Only 5%–10% of the filtered gentamicin is taken up and sequestered by the proximal tubule cells, where the aminoglycoside can achieve concentrations vastly exceeding the concurrent serum concentration. For many years, the proximal tubule has been considered the primary site of gentamicin-induced AKI and proximal tubular damage, mainly apoptosis, was frequently observed.^3,33,48 Interestingly, besides the observed predominant accumulation of gentamicin in proximal tubule cells, a significant intracellular accumulation of gentamicin in the CDs was previously reported.⁴⁹ How CDs accumulate gentamicin remains to be elucidated.

The kidney CD has been known for decades to be the key segment for water and salt transport and for regulating fluid balance. Its involvement in kidney injury has not been appreciated until recently.^30,50 Emerging studies have suggested that renal CD epithelial cells serve as a major source of inflammatory cytokines during AKI such as ischemia-reperfusion injury.^51,52 We and others have previously shown that disruption of the CD PC is “inflammatory” and results in significant damage to the overall kidney structure and function.^30,53,54 Most recently, we reported that integrin-linked kinase (ILK) deficiency in the CD promotes necroptosis. CD necroptosis resulted in massive inflammation and fibrosis of the kidney in ILK knockout mice.⁵⁵ This study highlights again the importance of the CD integrity to the well-being of the kidney. CD dysfunction, such as ILK deficiency, is sufficient to induce prominent renal failure. Our study reveals that significant necroptosis occurred in CD 7 days after gentamicin treatment. This finding implies that CD injury, which has been overlooked in the past, may in fact be an important event that mediates massive interstitial inflammation and fibrogenesis in gentamicin-induced AKI.

Our data demonstrates the critical involvement of CD necroptosis in gentamicin-induced AKI. Manipulating the level of expression, activity, or trafficking of an individual component of the “necrosome,” including RIPK1, RIPK3, and MLKL, have all been shown to affect necroptosis in cells or animals.^20,23,24 Besides necroptosis, another caspase-independent, regulated necrosis, ferroptosis is also known to play an important role in mediating tissue injury.^56,57 Ferroptosis is characterized by increased lipid peroxidation resulting from lack of activity of the glutathione peroxidase 4 that requires glutathione to function.^58,59 We examined the expression level of two key ferroptosis regulators GPX4 and SLC7A11 using quantitative PCR, which did not show any significant changes before and after gentamicin treatment in mouse kidney. We further tested the effect of a ferroptosis inhibitor, Fer-1, in our gentamicin-induced cytotoxicity assay. Fer-1 did not show any protective effects against gentamicin-induced cell death in vitro. Although our preliminary data do not support a major role of ferroptosis in gentamicin-induced cell injury, we cannot exclude confidently the involvement of ferroptosis in gentamicin-induced AKI in vivo at this point. It would be important and necessary to examine systemically the involvement of ferroptosis as well as other programmed cell death pathways in gentamicin-induced tubular injury.

Disclosures

J. Yuan has a patent “Necrostatins,” with royalties paid to Denali Therapeutics. All remaining authors have nothing to disclose.

Funding

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01-DK-096015 and R21-DK-092619, NephCure Foundation, an American Society of Nephrology Gottschalk research grant, the S&R Foundation Ryuji Ueno Award, American Heart Association Transformative Award, and MGH Executive Committee on Research support (to H.A. Lu). The Microscopy Core facility of the MGH Program in Membrane Biology receives additional support from the Boston Area Diabetes and Endocrinology Research Center through NIDDK grant DK-57521 and from the Center for the Study of Inflammatory Bowel Disease through NIDDK grant DK43351.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019020204/-/DCSupplemental.

Supplemental Table 1. List of primers used for quantitative real time PCR.

Supplemental Figure 1. Gentamicin causes kidney tubular injury and inflammation with mild proteinuria.

Supplemental Figure 2. Gentamicin induces necroptosis other than ferroptosis in cultured renal tubular cells and kidney.

Supplemental Figure 3. Nec-1 alleviated gentamicin-induced inflammatory cell infiltration.