Cellular and molecular mechanisms of kidney injury

HK2 cells were obtained from American Tissue and Cell Center (ATCC, CRL-2190) and grown as a monolayer in DMEM/F12 (Gibco) supplemented with 10% FBS (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and ITS (Sigma-Aldrich). Primary antibodies for dsRNA mAb J2 (Scions, 10010500, 1:200), PNPT1 (Abcam, ab96176, 1:200/2000), AQP1 (Abcam, 168387, 1:200), synaptopodin (Abcam, ab224491, 1:100), podocin (Sigma-Aldrich, P0372, 1:100), PKR (Abcam, ab32506, 1:1000), p-PKR (Abcam, ab32036, 1:1000), eIF2α (CST, 5324 S, 1:1000), p-eIF2α (CST, 3398 S, 1:1000), ATF4 (Abcam, ab184909), COX IV (Abcam, ab14744, 1:1000), LaminB1 (Abcam, ab65986, 1:1000), and α-tubulin (Proteintech, 11224-1-AP, 1:2000) were used for immunofluorescence and western blot analysis; Secondary antibodies including goat anti-mouse Alexa Fluor 488 (Invitrogen, A11001, 1:1000), donkey anti-rabbit Alexa Fluor 594 (Invitrogen, A21207, 1:1000), donkey anti-rabbit Alexa Fluor 488 (Invitrogen, A21206, 1:1000), goat anti-mouse Alexa Fluor 594 (Invitrogen, A11005, 1:1000), goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005, 1:1000) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2004, 1:1000) were used correspondingly. DAPI was purchased from Santa Cruz Biotechnology (sc-3598, 1:1000). TGFβ (R&D Systems, 7754), LPS (Sigma-Aldrich, L2630), HG (Sigma-Aldrich, G7021) were used. Digitonin (Sigma-Aldrich, D141) were used for membrane permeabilization. KIM-1 (Abcam, ab213477), NGAL (Abcam, ab118901), β₂-MG (Abcam, ab223590) ELISA quantitation kits, creatinine kit (Sigma-Aldrich, MAK080), calcium assay kit (Sigma-Aldrich, MAK022) and phosphorus microplate assay kit (Absin, 580107) were purchased for renal tubular injury assessment. Lipofectamine RNAi MAX (Invitrogen, 13778150) were used for transfection. PKR inhibitor (C16) was obtained from Sigma-Aldrich (I9785) and dissolved in DMSO prior to experiment. IMT1 was obtained from MCE (HY-134539) and dissolved in DMSO prior to experiment. FITC-Annexin V Apoptosis Detection Kit (Invitrogen, V13241) and TUNEL Bright-Green apoptosis detection kit (Vazyme, A112) were used for apoptosis analysis. Collagenase D (1 mg/mL, C6885), protease (1 mg/mL, P6911) and DNaseI (1 U/mL, D5025) were purchased from Sigma-Aldrich. Click-iT® Plus OPP Protein Synthesis Assay Kits were purchased from Life Technologies (C10456). PierceTM Classic IP Kit were purchased from Thermo Fisher Scientific (26146). Ribo^TM Fluorescent In Situ Hybridization Kit were purchased from Ribo (C10910). PNPT1 AAV were purchased from OBiO (H131559). siPNPT1 plasmid (siG000087178A-1-5) and siPKR plasmid (siB161220105622-1-5) were purchased from Ribo.