Af1817

Western blotting was performed as previously described (21 (link)). Mouse kidney and cultured cells were lysed in RIPA lysis buffer. Anti-GAPDH (1:2000 dilution, CW0100M, CWBio, China), anti-cleaved caspase 3 (1:1000 dilution, ab32503, Abcam, Cambridge, UK), anti-Bcl2 (1:1000 dilution, cst2870, Cell Signaling Technology, USA), anti-SIRT1 (1:1000 dilution, GT1189, Genetex, China),anti-KIM-1(0.25ug/ml, AF1817, RD systems, Canada), anti-PGC-1a(1:1000, # 2178, cell signaling technology, USA) and anti-Bax (1:1000 dilution, ab193349, Abcam Cambridge, UK) antibodies were used in this study. Experiments were repeated three times.

The kidney sections were ground using a homogenizer or the cells were scratched and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with proteinase inhibitor (Roche, Switzerland) for 30 min on ice. Protein concentration was quantified using the Bicinchoninic acid assay (Beyotime, Shanghai, China). Protein samples (50 μg) were used to perform immunoblotting as described previously^{42 (link)}. The specific primary antibodies used were: anti-GSDME (ab215191, Abcam, 1:1000), anti-caspase-3 (9662, CST, 1:1000), anti-cleaved caspase-3 (9661, CST, 1:1000), phospho-NF-κB p65 (3033, CST, 1:1000), NGAL (ab63929, Abcam, 1:1000), KIM-1 (AF1817, R&D systems, 1:1000), and anti-GAPDH (5174, CST, 1:10000). Peroxidase-conjugated goat anti-rabbit secondary antibodies (A0277, Beyotime, Shanghai, China) were used for immunoblotting.

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from human and mouse using antibodies against RXRα (ET7108-99, Huabio, 1:200 dilution), α-SMA (ab5694, Abcam, 1:100 dilution), Collagne-1 (ab34710, Abcam, 1:800 dilution), and Fibronectin (F3648, Sigma, 1:1000 dilution). Briefly, deparaffinized and rehydrated sections were boiled in citrate antigen retrieval solution for antigen retrieval. After incubation with 10% goat serum, sections were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ZLI-9018, ZSGB-BIO, 1:20 dilution). DAB-positive staining was assessed by two experienced pathologists in a blinded manner and evaluated by H score as described previously^{40 (link)}. For immunofluorescence, deparaffinized and rehydrated sections were blocked and incubated with antibodies against KIM-1 (AF1817, R&D System, 1:200 dilution) overnight at 4 °C, followed by incubation with Alexa Fluor 555-conjugated secondary antibodies (Invitrogen, 2273776, 1:1000 dilution) and fluorescein-LTL (Vector Lab, FL-1321, 1:1000 dilution). The tissue slides were imaged with a fluorescence microscope (DMi8, Leica).