2.2. DPP-4 Activity Assay

DPP-4 activity was determined in plasma and tissues as previously described [10]. Briefly, 0–40 μM of AMC (7-amino-4-methylcoumarin) standard was loaded into each well and read fluorometrically at Em/Ex = 360/465 nm, generating a linear standard curve. Next, 50 μL of plasma sample was added to each of the 96 wells, followed by the addition of 50 μL substrate solution (final concentrations 0.1 M HEPES, 50 μM Gly-Pro-AMC, and 50 μg/mL BSA) per well. Free AMC, which was released by DPP-4 activity, was quantitated by fluorometric measurements every 25 s for 300 s using a Spectramax GEMINI XPS microplate reader (Molecular Devices, Sunnyvale, CA, USA; Em/Ex = 360/465 nm, target temperature = 25 °C). To analyze DPP-4 activity in renal tissue, frozen tissue (10 mg) was homogenized in cold assay buffer (25 mM Tris-HCl, 140 mM NaCl, 10 mM KCl, pH 7.5, 0.1% BSA) and spun by centrifugation at 20,000× g for 20 min at 4 °C. DPP-4 activity in the lysates was measured as described above. DPP-4 activity in each tissue sample was expressed as the amount of cleaved AMC per minute per gram of tissue (μM/min/g tissue).