Reagents. We purchased the following reagents as follows: BV6 (B4653, ApexBio), cisplatin (AG-CR1-3590, Adipogen), GSK’872 (530389, Merck), IFNb (12401-1, PBL-Assay Science), LPS (434, List Labs), nigericin (AG-CN2-0020, Adipogen), Murine TNF (34-8321, eBioscience), zVAD (3188-v, Peptide Institute), and 5z7Ox (499610, Millipore). The following antibodies were used in this study: anti-actin (A2066, Sigma-Aldrich, diluted 1:1000), anti-cleaved caspase 3 (9661, Cell Signaling Technology, diluted 1:1000), phycoerythrin (PE) cyanine-7–conjugated anti-CD11b (101215, Biolegend, diluted 1:100), PE-conjugated anti-F4/80 (50-4801, TOMBO biosciences, diluted 1:100), anti-green fluorescent protein (GFP) (sc-8334, Santa Cruz Biotechnology, diluted 1:5000), anti-MLKL (3H1, Millipore, diluted 1:1000), anti-phospho-RIPK3 (57220, Cell Signaling Technology, diluted 1:1000), horseradish peroxidase (HRP)–conjugated donkey anti-rat IgG (712-035-153, Jackson ImmunoResearch Laboratories, Inc, 1:20000), HRP–conjugated donkey anti-rabbit IgG (NA934, GE Healthcare, 1:20000), biotin-conjugated donkey anti-rabbit IgG (E0432, Dako, 1:200), and Streptavidin-HRP (P0397, Dako, 1:300).
Cell culture. Primary MEFs (pMEFs) were prepared from SMART Tg mice at E14.5 after coitus with a standard method. We prepared immortalized wild-type and Mlkl-/- MEFs (iMEFs) described previously 22. MEFs were maintained in DMEM medium containing 10% fetal bovine serum (FBS).
Mice.Ripk3-/- mice45 (provided by Genentech Inc.) were described previously. C57BL/6 mice (Sankyo Lab Service) were housed in a specific pathogen-free facility and received a routine chow diet and water ad libitum. All animal experiments were performed according to the guidelines approved by the Institutional Animal Experiments Committee of Toho University School of Medicine (approval number: 21-54-400), Kyoto Graduate School of Medicine (approval number: Medkyo 21562), and the RIKEN Kobe branch (approval number: QA2013-04-11).
Generation of SMART Tg mice. SMART Tg mice were generated by microinjecting Tol2 mRNA and the pT2KXIG-mSMART vector into the cytoplasm of fertilized eggs from C57BL/6 mice, as described previously26. Eight- to twelve-week-old male and female mice were used for the in vivo imaging.
Western blotting. Murine tissues were homogenized with a Polytron (Kinematica, Inc.) and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). After centrifugation, cell lysates were subjected to SDS polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (IPVH 00010, Millipore). The membranes were immunoblotted with the indicated antibodies and developed with Super Signal West Dura Extended Duration Substrate (34076, Thermo Scientific). The signals were analyzed with an Amersham Imager 600 (GE Healthcare Life Sciences).
Flow cytometry. Cells were stained with anti-CD11b and anti-F4/80 antibodies in flow cytometry staining buffer (eBioscience). The prepared cells were gated on forward and side scatter to identify the lymphocyte population, and then discriminating doublets. Cells were analyzed with a BD FACSCant II flow cytometer (BD Biosciences) and FlowJo software (BD Biosciences).
Cell death assay. Macrophages were plated onto 96-well plates and cultured for 12 h in RPMI medium containing 10% FBS. Macrophages were stimulated with the IAP antagonist, BV6 (1 µM), in the presence of the apoptosis inhibitor, zVAD (20 µM), the RIPK3 inhibitor, GSK’872 (5 µM), or both, as indicated, for the times indicated. To induce pyroptosis, macrophages were pretreated with LPS (1 ng/mL) for 4 h, then stimulated with nigericin (10 mM) for the indicated times. Primary MEFs were untreated or pretreated with IFNb (2000 IU/mL) for 24 h, then stimulated with TNF (10 ng/mL) and BV6 in the presence of zVAD (20 mM) or GSK’872 (5 mM), as indicated, for 18 h. The concentrations of LDH released from cells were determined with a Cytotoxicity Detection Kit (Roche), as described previously 46.
In some experiments, macrophages were pretreated with LPS (1 ng/mL) for 4 h, and then stimulated with 5z7Ox (125 nM) in the presence of zVAD (20 mM), GSK’872 (5 mM), or both, as indicated, for 4 h. Cell viability was determined with a water-soluble tetrazolium salts assay (Cell Counting kit-8, Dojindo Molecular Technologies).
FRET analysis in vitro. FRET analysis was performed as previously described. Briefly, FRET signals were imaged with a DeltaVision microscope system (GE healthcare) built on an Olympus IX-71 inverted microscope base equipped with a Photometric Coolsnap HQ2 CCD camera and a 60×/NA1.516 PlanApo oil immersion lens (Olympus). For live-cell imaging FRET sensors, cells were seeded on gelatin-coated CELLview Cell Culture Dishes (Greiner Bio-One) and maintained in an incubator at 37°C with 5% CO2. For imaging, cells were observed with a Blue excitation filter (400–454 nm), two emission filters (blue-green, 463–487 nm for ECFP; yellow-green, 537–559 nm for Ypet), and a C-Y-m polychronic mirror. The FRET emission ratio (FRET/CFP) was calculated with SoftWoRx (Applied Precision Inc) by dividing the excitation at 436 nm and emission at 560 nm (FRET) by the excitation at 436 nm and emission at 470 nm (CFP). For statistical analyses, the obtained images were analyzed with ImageJ and MetaMorph software. The ΔFRET/CFP ratios were calculated by subtracting the FRET/CFP ratio at time 0 from the FRET/CFP ratio at the indicated times.
Isolation of peritoneal exudate macrophages. To isolate peritoneal exudate macrophages, 6- to 8-week-old mice of the indicated genotypes were intraperitoneally injected with 2.5 ml 3% thioglycollate (T9032, Sigma). On day 4 after the thioglycollate injection, anesthetized mice were intraperitoneally injected with ice-cold PBS; then, the peritoneal cells were harvested when the PBS was recovered. This procedure was repeated twice. Harvested cells were placed in plates with RPMI medium. After removing non-adherent cells, the remaining cells were primarily peritoneal macrophages. Approximately 80% of these cells were positively stained with CD11b and F4/80 antibodies.
Injection of cisplatin into mice. To produce cisplatin-induced kidney injuries, 8- to 12-week-old mice of the indicated genotypes were injected with cisplatin (20 mg/kg). Injected mice were sacrificed at the indicated times, and sera and kidney tissues were collected for subsequent analyses.
Histological, immunohistochemical, and immunofluorescence analyses. Tissues were fixed in 10% formalin and embedded in paraffin blocks. Paraffin-embedded kidney sections were used for hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence analyses. For immunohistochemistry, paraffin-embedded sections were treated with Instant Citrate Buffer Solution (RM-102C, LSI Medicine) for antigen retrieval. Next, tissue sections were stained with anti-CC3 antibody, followed by the secondary antibody, biotin-conjugated anti-rabbit antibody. Streptavidin-HRP was added for visualization. Images were acquired with an all-in-one microscope (BZ-X700, Keyence) and analyzed with a BZ-X Analyzer (Keyence). CC3+ cells were automatically counted in three randomly selected high-power fields (original magnification, ×40) per kidney, with a Hybrid Cell Count (Keyence).
For the immunofluorescence analyses, tissue sections were preincubated with MaxBlock™ Autofluorescence Reducing Kit (MaxVision Biosciences), according to the manufacturer’s instructions. Next, tissue sections were stained with anti-pRIPK3 antibody, followed by visualization with the tyramide signal amplification method, according to the manufacturer’s instructions (NEL741001KT, Kiko-tech). Images were acquired with an LSM 880 (Zeiss). The images were processed and analyzed with ZEN software (Zeiss) and an image-processing package, Fiji (https://fiji.sc/). pRIPK3+ cells were counted manually.
Measurement of blood urea nitrogen and creatinine. After the cisplatin injection, serum samples were collected on days 0, 1, 2, and 3. Serum creatinine (measured with an enzymatic method) and blood urea nitrogen (measured with the urease-glutamate dehydrogenase method) were measured by Oriental Yeast.
Imaging cisplatin-induced kidney injury. Living mice were observed with an FV1200MPE-BX61WI upright microscope (Olympus) equipped with an XLPLN 25XW-MP 25X/1.05 water‐immersion objective lens (Olympus). The microscope was equipped with an InSight DeepSee Ultrafast laser (0.95 Watt, 900 nm; Spectra-Physics, Mountain View, CA). The scan speed was set at 2 µs/pixel. The excitation wavelength for CFP was 840 nm. Fluorescence images were acquired with the following filters and mirrors: an infrared-cut filter (BA685RIF-3); two dichroic mirrors (DM505 and DM570); and three emission filters, including: an FF01-425/30 (Semrock, Rochester, NY), for the second harmonic generation; a BA460-500 (Olympus) for CFP; and a BA520-560 (Olympus) for FRET. The microscope was also equipped with a two-channel GaAsP detector unit and two multi-alkali detectors. FluoView software (Olympus) was used to control the microscope and acquire images. Acquired images were saved in the multilayer 12-bit tagged image file format and processed and analyzed with Metamorph software, as described previously 47.
Intravital mouse imaging was performed essentially as described previously 47. Briefly, two days before imaging, cisplatin was injected intravenously. To observe the kidney, the mouse was placed in the prone position on an electric heat pad maintained at 37℃. A 1-cm incision was made in the skin of the lower back and underlying peritoneum to expose approximately 0.25 cm2 of tissue. The exposed tissue was imaged with an aspiration fixation system. The obtained images were analyzed by Metamorph. The FRET/CFP ratios of relevant areas were calculated with ImageJ software. Then, the averaged FRET/CFP ratios were calculated for each proximal tubule.
Statistical analysis. Statistical analyses were performed with the unpaired two-tailed Student’s t-test or one-way or two-way ANOVA with Dunnett’s, Sidak’s, or Tukey’s multiple comparison test, as appropriate. P-values < 0.05 were considered statistically significant.