Mrt10

Serum was collected monthly from the control and treated rats, and inflammatory cytokines were assessed by enzyme-linked immunosorbent assays (ELISAs) using rat ELISA kits according to the manufacturers' protocols. For rat TNF-α and TNF receptor 1 (TNFR1), catalog #RTA00 and MRT10 from R & D Systems (Minneapolis, MN) were used, respectively; for interleukin-8 (IL-8) catalog #R6417 from Biotang Inc. (Waltham, MA); for the S100A8/A9 complex catalog #MBS036705 from MyBiosource.com (San Diego CA); and for C-reactive protein (CRP) catalog #RAB0097 from Sigma-Aldrich Inc. (St. Louis, MO). All serum assays were performed in triplicate.

Body weight, food intake, water intake, and hemoglobin A1c (HbA1c) were measured at 6 and 15 weeks of age. The mice were placed in metabolic cages for 24 h for albumin/creatinine ratio measurement using DCA 2000 microalbumin–creatinine immunoassay cartridges with a DCA Vantage Analyzer (Siemens Healthcare, Erlangen, Germany). Blood HbA1c levels were analyzed using DCA 2000 HbA1c immunoassay cassette and DCA Vantage Analyzer (Siemens Healthcare). Serum tumor necrosis factor 1 (TNFR1) and TNFR2 levels were measured by an ELISA kit (R&D Systems, Minneapolis, MN, USA, MRT10, MRT20), according to the manufacturer's instructions. At least three to six blood pressure measurements were taken per session in 15‐week‐old mice using a noninvasive tail sleeve and pulse transducer system (BP‐98A; Softron, Tokyo, Japan), which were preheated for 10 min at 38 °C. A standard deviation of < 10.0 was used for blood pressure levels.