Anti kim 1

Protein was isolated from kidney tissues and loaded onto a gradient polyacrylamide gel. The resolved proteins were transferred onto a nitrocellulose membrane and the membrane was incubated with the following primary antibodies: anti-Kim-1 (Abcam), anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-Bax (Santa Cruz Biotechnology), anti-cleaved poly(ADP-ribose) polymerase-1 (PARP1; Cell Signaling), anti-acetyl-p53 (Lys379; Cell signaling), anti-p53 (Cell Signaling), anti-interleukin-6 (IL-6; Abcam), anti-tumor necrosis factor-α (TNF-α; Abcam), anti-acetyl-NF-κB p65 (Lys310; Cell Signaling), anti-NF-κB p65 (Cell Signaling), and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling) (Cell Signaling) antibody. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies, and signals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA). The protein expression levels were normalized against GAPDH.

Western blotting was performed as previously described [12 (link)]. Briefly, total protein from kidney tissue and cells were extracted by lysis buffer (Thermo Scientific, Waltham, MA). Protein concentrations were detected using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Individual immunoblots were probed with primary antibodies as followed: Anti-GAPDH (CW0100M; Cwbio, Beijing, China), anti-KIM-1 (ab190696, Abcam), anti-p-p65 (#3033, CST), anti-Bax (#2772, CST), and anti-cleaved caspase-3 (#9661, CST).

For preparing the protein sample, HK-2 cells and kidney tissues were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer, and the supernatants were collected after centrifugation at 13,000 g at 4°C for 15 min. Protein concentration was quantified using the BCA Protein Assay (K813-5000-1; BioVision). Proteins were separated with 8%–15% SDS-PAGE and blotted onto PVDF microporous membranes (IPVH00010; Millipore Sigma). The membranes were then blocked with 5% milk at room temperature for 1 h and incubated with primary antibodies at 4°C overnight, followed by incubating with HRP-conjugated secondary antibodies for 1 h at room temperature. The primary antibodies are as follows: anti-mKlotho (AF1819; R&D Systems), anti-PARP (#9532; Cell Signaling Technology), anti-p53 (#2524S; Cell Signaling Technology), anti-FADD (sc-271748; Santa Cruze Biotechnology), anti-cacspase-3 (#9622; Cell Signaling Technology), anti-Coronavirus nucleocapsid (sc-66012; Santa Cruze Biotechnology), anti-Flag (F1804; Sigma-Aidrich), anti-KIM-1 (Ab47635; Abcam), anti-Lipocalin-2/NGAL (ab63929; Abcam), anti-GAPDH (Z1208; Ray antibody Biotechnology). The protein bands were quantified by ImageJ software (NIH) and normalized to the loading controls.

Protein from kidney tissues was extracted with RIPA buffer (Sigma‐Aldrich, Hamburg, Germany) in the presence of Protease and Phosphatase Inhibitor Mini Tablets (Thermo Fisher, Rockford, IL, USA). Equal amounts of protein were loaded onto sodium dodecyl sulfate polyacrylamide gels for electrophoresis and western blotting. The blots were probed with the following primary antibodies at 4°C overnight: anti‐KIM1, anti‐MDM2 (Abcam, Cambridge, UK), anti‐phospho‐MDM2, anti‐p53, anti‐phospho‐p53, anti‐NFκβ, anti‐phospho‐NFκβ, anti‐Iκb, anti‐phospo‐Iκb and β‐actin (Cell Signaling Technology, Danvers, MA, USA). After washing the primary antibody, the membranes were incubated with peroxidase‐conjugated secondary antibody. The bands were visualized by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).

Kidney protein samples were extracted using lysis buffer (CelLytic™ MT, Sigma-Aldrich, St Louis, MI, USA) and centrifuged at 13,000 rpm, at 4 °C, for 10 min after incubation on ice for 30 min. The supernatant was collected and the protein concentration was measured using a Bio-Rad Bradford kit (Bio-Rad Laboratories, Hercules, CA, USA) at 595 nm using a spectrophotometer. The samples were boiled for 10 min, and equal volumes were loaded on precast gradient polyacrylamide gels (Bolt™ 4-12% Bis-Tris Plus Gels; Thermo Fisher Scientific, Waltham, MA, USA) before being transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). The membrane was blocked for 2 h at room temperature in 5% BSA and incubated with primary antibodies (1:1000) overnight, at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution) were used in this study. The signal intensity was detected using an image analyzer (ChemiDoc™XRS+, Bio-Rad Laboratories) and quantified using the Image Lab software (Bio-Rad Laboratories). The primary antibodies used in this study were: anti-LC3B, anti-Beclin1, and anti-α tubulin (Cell Signaling Technology, Inc.); anti-fibronectin, anti-TNF-α, anti-Kim-1, and anti-collagen I (Abcam); anti-IL-1β, anti-HIF-1α, and anti-NGAL (Santa Cruz Biotechnology lnc., USA); anti-TFEB (Novus Bio, Littleton, CO, USA).

Expression levels of Tamm-Horsfall protein (THP), pan-keratin (Ker), NGAL, and KIM-1 were measured using immunofluorescence. Briefly, after a 2-week differentiation period, 3D kidney organoids (3D-embedded) in PBS were pipetted very gently to separate them from the Matrigel matrix for whole organoid stain. Whole organoids seeded (37°C, 16 h) onto an 8-chamber cell-culture slide were fixed (10 min) in 4% paraformaldehyde solution and washed in PBS. Cells were permeated (5 min) in 0.5% Triton X-100/PBS, blocked (20 min) with 5% BSA/PBS, and incubated (4°C, 16 h) in diluted primary antibodies: FITC-conjugated anti-THP (Cedarlane Laboratories, Burlington, NC, USA), anti-NGAL (Abcam, Cambridge, UK), anti-KIM-1 (Abcam) and anti-pan-keratin (Cell Signaling Technology, Danvers, MA, USA). Finally, PBS-washed cells were exposed (25° C, 1 h) to secondary antibodies tagged with Alexa Fluor 488 or 594 (1:200; Invitrogen [Thermo Fisher], Waltham, MA, USA). Images were captured using a confocal microscope (LSM 700 META; Carl Zeiss AG, Oberkochen, Germany) and analyzed using proprietary software (LSM Image Browser).

Ibudilast and FA were purchased from Meilun Biotechnology Co. (Dalian, Liaoning, China). We used the following antibodies to assess protein expression: anti–KIM-1, anti-NLRP3, anti–caspase-1, anti-GSDMD, and anti–β-actin antibodies from Abcam (Cambridge, MA, United States of America); anti–p-p65, anti-p65, anti–p-IκBα, anti-IκBα, anti–p-ERK, anti-ERK, anti–p-p38, anti-p38, anti–p-JNK, anti-JNK, anti-F4/80, and anti–IL-1β antibodies from Cell Signaling (Danvers, MA, United States of America); and anti–IL-18, anti–TNF-α, and anti-TLR4 antibodies from Proteintech (Wuhan, China).