SGLT2 inhibition and inflammasome pathway and diabetic nephropathy
Description
This study investigated the effects of sodium glucose cotransport 2 (SGLT2) inhibition on cardiac redox homeostasis and inflammation in an experimental model of DN. Male C57BL/6J mice were fed either standard chow (SC) or high-fat diet (HFD) for 16 weeks, with the HFD animals receiving streptozotocin (40 mg/kg) at week 8. At week 17, mice were divided into four groups: Sham, Sham+SGLT2i, DN, and DN+SGLT2i (empagliflozin 35 mg/kg for 8 weeks), and their cardiac tissues were analyzed for oxidative stress markers and inflammatory mediators.
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Steps to reproduce
1. Experimental Model and Treatment - Use male C57BL/6J mice (20–25 g, ~60 days old). - Induce diabetic nephropathy (DN) with a high-fat diet (16 weeks) and a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg; Sigma-Aldrich) at week 8 after a 6-hour fast. - Include only mice with blood glucose levels >180 mg/dL after 72 hours. - At week 17, allocate mice into four groups: Sham, Sham+SGLT2i, DN, and DN+SGLT2i. - Treat the SGLT2i groups with empagliflozin (Jardiance®) at 35 mg/kg/day via oral gavage for 8 weeks. 2. Tissue Collection and Sample Preparation - Euthanize animals by cervical dislocation. - Excise ventricular cardiac tissue, divide into equal aliquots, snap-freeze in liquid nitrogen, and store at −80 °C. - Prepare homogenates using RIPA buffer with protease inhibitors. - Quantify protein concentration using the Bradford assay. 3. Biochemical Assays A. Oxidative Stress Markers - Hydrogen Peroxide (H₂O₂): Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen A22188). - Catalase (CAT) and Glutathione Peroxidase (GPx): Follow Weydert & Cullen (2010). - Protein Carbonyls: DNPH-based assay (Dalle-Donne et al., 2003). B. ELISA (In-House) - IL-1β: Capture antibody Invitrogen PA5-79485; TMB substrate; BSA blocking; PBS-Tween washes. - 8-OHdG: Capture antibody Santa Cruz SC-393871; biotinylated detection antibody; TMB incubation. C. Western Blot - Primary antibodies: ASC (Abcam ab70627), Caspase-1 (Invitrogen PA5-8736), 4-HNE (Bioss BS-6313R), β-actin (Invitrogen MA515452). - Secondary antibodies: HRP-conjugated IgG. - Detection: ECL chemiluminescence; ImageJ for densitometry. 5. Order of Operations for Data Reanalysis - Normalize biochemical assays to protein content. - Generate ELISA standard curves and calculate concentrations. - Quantify Western blot bands and normalize to β-actin. - Group data by experimental condition. 7. Notes on Reproducibility - Maintain stable environmental conditions (25°C; 12h light/dark cycle). - Use consistent antibody lots when possible. - Standardize ELISA incubation times and Western blot exposure conditions. - Freeze tissue immediately after collection to prevent degradation.
Institutions
- Pontificia Universidade Catolica do Parana