Fig 1: Gene deficiency hindered the NLRP3 inflammasome activation of renal tubular epithelial cells of mice. Primary RTECs from NLRP3-/- and control mice were stimulated with chemical hemes for 4 h, following (A–C) analysis. A, Western blot analysis of indicated protein in total lysates (CL) and supernatants (SN) of NLRP3-/-RTECs and control mice primary RTECs. B, Electron microscopy scanning on NLRP3-/-RTECs and control mice primary RTECs (Magnification: the upper = 1000×; the lower = 1000×). The lower panels were enlarged from the white box of upper panel. C, The observation and analysis of nuclei (DAPI, blue) and GSDMD (green) foci of NLRP3-/- RTECs and control mice RTECs by laser confocal microscope (bar = 50µm). D, The animal model of hemolysis was established by the injection of human blood plasma. E, The urine was collected from each group and analyzed free hemoglobin. F–G, The analysis of blood urea nitrogen (BUN) and the creatinine (CREA). H, The H&E staining of kidney section from NLRP3-/- and control hemolysis mice kidney section (bar = 200 µm), and the tubular cell necrosis scores. The lower panel was enlarged from the green box of upper panel to show the tubular damage region. I, The detection of TIM-1 and NGAL by laser confocal microscope and quantity analysis of containing TIM-1+ foci and NGAL+foci in NLRP3-/- and control hemolysis mice kidney section (bar = 200µm). Each data represented data five mice per group. Data shown represent the mean ± SD from at least triplicate measurements. * P < .05; ** P < .01; *** P < .001
Fig 2: Heme injured renal tubular epithelial cells through NLRP3 inflammasome activation in vitro. A, Primary RTECs were stimulated for 4 h with 50µM chemical hemes. Western blot analysis for NLRP3, pro-caspase1, caspase1p20, pro-IL-1ß, IL-1ß, and GSDMD in RTECs after challenge. Total RTECs lysates (CL) and supernatants (SN) using indicated antibodies were detected after stimulation. B, ELISA analysis of IL-1ß in primary RTECs supernatants. C, HK-2 cells were stimulated for 4 h with 50 µM chemical hemes. Western blot analysis for NLRP3, pro-caspase1, caspase1p20, pro-IL-1ß, IL-1ß, and GSDMD in HK-2 cells after challenge. Total HK-2 lysates (CL) and supernatants (SN) using indicated antibodies were detected after stimulation. D, ELISA analysis of IL-1ß in HK-2 cells supernatants. E and F, Scanning electron microscopy observation of primary RTECs and HK-2 cells (Magnification: the upper = 1000×; the lower = 10 000×) pyroptotic pore numbers after 50 µM chemical hemes treatment. The lower panel was enlarged from the white marked area of upper panel. G and H, The observation of nuclei (DAPI, blue) and GSDMD (red) foci of RTECs and HK-2 cells by laser confocal microscope (bar = 50µm) and quantity of primary RTECs containing GSDMD+ foci. The rightmost panel was enlarged from the white box of neighbor panel. Data shown represent the mean ± SD from at least triplicate measurements. * P < .05; ** P < .01; *** P < .001
Fig 3: NLRP3 inflammasome was activated by hemes in the murine models of hemolysis. The animal model of hemolysis was established by transfused with human plasma as mentioned above, then followed by (A–D, I–J). A, Primary RTECs were isolated from transfusion mice and cultured for 2 h at 37°C. Proteins were prepared from the mixture of cells and the supernatant to perform the western blot of NLRP3, ASC, Caspase1, IL-1ß, and GSDMD. B–D, Densitometry analyses of caspase-1, GSDMD, and IL-1ß based on (A). The animal model of hemolysis was established by injection of mice heme from destroyed RBCs, followed by (E–H). E. Western blot analysis for indicated proteins in primary RTECs from mice. F-H, Densitometry analyses of IL-1ß, Caspase-1, and GSDMD based on (E). I, The observation of GSDMD (red) foci of RTECs from the animal models of hemolysis transfused with human plasma by laser confocal microscope. J, Quantity of RTECs containing GSDMD+ foci, according to (I). Each data represented five mice per group, and quantity data are shown as mean ± SD. * P < .05; ** P < .01; *** P < .001
Fig 4: The activation of NLRP3 inflammasome due to heme was inhibited by 66PR and MCC950 compound in renal tubular epithelial cells. A,B, The 66PR compound was synthesized and linked with biotin. Then it was used to treat HK-2 cells, and to detect the binding with NLRP3 by co-Immunoprecipitation. C, Western blot analysis of NLRP3, Caspase-1, GSDMD, and IL-1ß in total lysates (CL) and supernatants (SN) from primary RTECs after inhibitor treatment. D–F, Densitometry analysis ofcaspase-1, IL-1ß, and GSDMD based on (C). G, ELISA analyses of IL-1ß release from primary RTECs supernatant after inhibitor administration. H, The observation of primary RTECs under light microscope after inhibitor treatment (bar = 50µm). I, The observation and analysis of nuclei (DAPI, blue) and GSDMD (red) foci of RTECs after inhibitor treatment by laser confocal microscope (bar = 50 µm). The rightmost panel was enlarged from the white box of neighbor panel. Fluorescence intensity (%) was the index of fluorescence, obtained by Image J software. J,K, The detection of TIM-1 and NGAL by laser confocal microscope and quantity of primary RTECs containing TIM-1+ foci and NGAL+foci after inhibitor treatment (bar = 50µm). The rightmost panel was enlarged from the white box of neighbor panel. Results are representative of five independent experiments. Data are shown as mean ± SD. * P < .05; ** P < .01; *** P < .001
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