Ace color

Plasma concentrations of triglyceride, cholesterol, phospholipid, FFA, and glucose were measured with commercial assay kits (Triglyceride E test Wako, Cholesterol E test Wako, Phospholipid C test Wako, NEFA C test Wako, and Glucose CII test Wako, Wako Pure Chemical Industries, Ltd., respectively). Plasma concentrations of adiponectin, insulin, and leptin were measured by enzyme-linked immunosorbent assay using a commercial kit (Mouse/Rat Adiponectin ELISA kit, Otsuka Pharmaceutical, Co. Ltd., Tokyo, Japan; Rat Insulin ELISA kit, Morinaga Institute of Biological Science, Kanagawa, Japan; and Mouse/Rat Leptin ELISA kit, Morinaga Institute of Biological Science, respectively). Plasma concentration of NOx was measured by detecting nitrogen dioxide and nitrogen trioxide, stable metabolites of nitric oxide, using a commercial kit (NO₂/NO₃ Assay kit-FX, Dojindo Laboratories, Kumamoto, Japan) after deproteinization of plasma by filtration using an Amicon Ultra Centrifugal Filter (Merck Millipore, Ltd., Carrigtwohill, County Cork, Ireland). Plasma specific activity of angiotensin converting enzyme (ACE) was measured with a commercial kit (ACE color Fujirebio, Inc., Tokyo, Japan). All measurements were performed in accordance with the manufacturer’s instructions. The primary outcomes were adiponectin, NOx, and ACE, and the secondary outcomes were the others.

Renal and serum enzymatic ACE activities were determined using a commercially available kit (ACE Color; Fujirebio, Hannover, Germany) according to the manufacturer’s instructions and adapted to 96-well plates. To determine the renal ACE activity, 10–36 mg of renal tissue was suspended in a lysis buffer [containing 50 mmol L HEPES, 0.5% Triton X-100, 0.025 mmol L ZnCl₂, 150 mmol L⁻¹ NaCl, 1 mmol L⁻¹ PMSF, 1 tablet EDTA-free protease inhibitor cocktail (cOmplete Mini, Roche, Basel, Switzerland); 10 µL Lysis buffer/1 mg tissue]. Samples were homogenized, incubated for 60 min at 4 °C, and centrifuged (15 min, 8000 g, room temperature). The supernatant was removed and assayed undiluted. To determine the serum ACE activity, samples were diluted (1:3).