Enzymatic assay kit

Plasma, urinary, and kidney cortical cytosolic (see below for ultracentrifugation; ‘Western blotting’) glucose concentrations were measured by the glucose oxidase method (Cayman Chemical, Ann Arbor, MI, USA). Glycated hemoglobin was measured in whole blood samples using an enzymatic assay kit (Crystal Chem Inc., Downers Grove, IL, USA). ELISA kits were used for the measurement of plasma insulin (Crystal Chem Inc.), urinary albumin (Bethyl laboratories, Montgomery, AL, USA), kidney injury molecule-1 (KIM-1; USCN Life Science Inc., Hubei, China), and neutrophil gelatinase-associated lipocalin (NGAL; R&D Systems, Minneapolis, MN, USA). Tissue levels of renin and AngII, and plasma levels of renin were determined via radioimmunoassay (Prosearch International, Malvern, VIC, Australia)50 (link).

The percentage of hemoglobin A_1c (HbA_1c) as a percentage of the total hemoglobin was analyzed by using an enzymatic assay kit (Crystal Chem, Elk Grove Village, IL). According to the manufacturer’s instructions, whole blood samples were lyzed in a commercial lysis buffer. Then, 25 µL of the lysate was incubated with 160 µL of a protease reaction mixture at 37 °C for 5 min, allowing the digestion and release of glycated valines from the beta chain of hemoglobin. The glycated valines are specific substrates for fructosyl valine oxidase; these reactions also produce hydrogen peroxide. Finally, the hydrogen peroxide produced was measured by the addition of 70 µL peroxidase and chromagen solution. After 3 min incubation at 37 °C, the optical density was recorded at 700 nm. The HbA_1c level in the sample was interpolated using a calibration curve, which was generated by plotting the mean absorbance values of the calibrators.