Kim 1

Serum creatinine was measured using an Atellica CH 930 Analyzer (Siemens Heathineers, Erlangen, Germany). To assess renal function, creatinine was also measured in urine samples according to the Jaffe principle. Urinary albumin concentration was measured using Hemocue^®Albumin 201 System (HemoCue AB, Ängelholm, Sweden). Blood glucose (GlucoMen^® Aero, A. Menarini diagnostics, Machelen, Belgium) and lactate levels (StatStrip Xpress, Nova Biomedical, Den Bosch, The Netherlands) were determined every 3 weeks until sacrifice. Plasma glucose was measured by using a colorimetric Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA). At sacrifice, blood β-ketone levels were measured (GlucoMen^® Aero 2K, A. Menarini diagnostics, Machelen, Belgium). Urinary NAG (Roche Diagnostics GmbH, Mannheim, Germany) and KIM-1 (MyBioSource, San Diego, CA, USA) were determined photometrically at the end of the study.

Serum creatinine was quantified by a purchased spectrophotometric assay kit (Bioassay Systems, CA, USA; cat# DICT-500) using an improved Jaffe method that utilizes picrate to form a red-colored complex with creatinine. Blood urea nitrogen (BUN) was evaluated spectrophotometrically using a colorimetric assay kit (Bioassay Systems; cat# DIUR-500) that depends on the modified Berthelot reaction. Serum levels of cystatin C (CUSABIO, MD, USA; cat# CSB-E08385r), kidney injury molecule-1 (KIM-1; MyBioSource, CA, USA; cat# MBS2702467), and IL-18 (LSBio, WA, USA; cat# LS-F2596) were determined using the corresponding rat ELISA kits. All experiments were processed according to the manufacturers’ instructions.

Tubular injury was semiquantitatively assessed in PAS-stained kidney tissues. To detect monocytes/macrophages or neutrophils, formalin-fixed, paraffin-embedded kidney tissues were stained with monoclonal antibodies against F4/80 (1:100; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G (1:200; eBioscience, San Diego, CA, USA). The mean number of positive cells per 8–10 high-power fields (HPFs) was compared. Tubular cell apoptosis was quantified by counting the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive epithelial cells in 8–10 HPFs (200×). NGAL (1:1000; Abcam, Cambridge, UK), anti-8-OHdG antibody [N45.1] (ab48508; ABcam, Cambridge, UK), SOD (1:800; MA1-105, ThermoFisher, Berkeley, MO, USA), catalase (1:400, PA5-29183, ThermoFisher, Waltham, MA, USA), KIM-1 (1:200, MBS2006453, MyBioSource, San Diego, CA, USA), and IGFBP7 (1:1000; Abcam, Cambridge, UK)-positive cells were semiquantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) [34 (link)].