13(S)HOTrE suplemention

1. Periodic acid–Schiff (PAS) staining Kidney tissues were fixed in 4% paraformaldehyde overnight, paraffin-embedded, and sectioned. Deparaffinized sections were treated with 1% periodic acid followed by Schiff’s reagent (Solarbio, China), then counterstained with hematoxylin and mounted. For quantification, ten random fields per section were imaged and analyzed using Image-Pro Plus software (Media Cybernetics, USA). 2. RNA extraction and quantitative real-time PCR Total RNA was isolated from kidney tissues or pTECs using Trizol (Invitrogen, USA) and verified for purity (A260/A280 1.8–2.0) via a NanoDrop 2000 (Thermo Fisher Scientific, USA). Subsequently, 1 μg of RNA was reverse-transcribed using a reverse transcription system (Vazyme, China). Real-time quantitative PCR (RT-qPCR) was performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, USA) using SYBR master mix (Vazyme, China). Relative mRNA expression was calculated via the 2-ΔΔCt method and normalized to GAPDH. 3. Measurement of GSH and GSSG Glutathione levels in kidney tissues and pTECs were quantified using a GSH/GSSG Assay Kit (Beyotime, S0053, China). Kidney samples were homogenized in RIPA buffer (Servicebio, China) and centrifuged (12,000 × g, 10 min, 4 °C), while pTECs were harvested and lysed following a PBS wash. Total glutathione and GSSG (determined using a GSH-masking reagent) were measured via the 5,5'-dithiobis (DTNB)-enzymatic recycling method at 412 nm. Reduced GSH was calculated by subtracting GSSG from the total. All data were normalized to protein concentrations determined by a bicinchoninic acid (BCA) kit (NCM Biotech, China) and expressed as nmol/mg protein or the GSH/GSSG ratio. 4. Measurement of COX‑2 enzyme activity and content of PGE₂ Kidney and pTEC lysates were sonicated and centrifuged (12,000×g, 10 min, 4 °C). COX-2 activity was determined using a fluorometric kit (Elabscience, China) by measuring ADHP fluorescence (Ex/Em: 535/587 nm) after incubation with arachidonic acid at 37 °C for 30 min. Results were calculated via an H₂O₂ standard and expressed as RFU/mg protein. PGE₂ concentrations were measured by an ELIASA kit (ELK Biotechnology, China). All data were normalized to total protein (BCA method) for tissues or reported as pg/mL for cell supernatants. 5. Immunofluorescence and TUNEL staining After citrate-buffered heat-induced antigen retrieval and serum blocking, sections were incubated overnight at 4 °C with primary antibodies: KIM-1 (R&D Systems, USA; 1:200), LTL (Vector Laboratories, USA; 1:100), GPX4 (Abclonal, China; 1:200) and GSS (Huabio, China; 1:200). Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI, Servicebio, China).