Urine albumin

Eight to twelve week-old male wild type and PAR-1 deficient mice (8 per group) were injected with streptozotocin (50 mg/kg body weight) for 5 consecutive days to induce diabetes. Eight age-matched, untreated wild type and PAR-1 deficient mice served as a non-diabetic control groups. Three months after streptozotocin injections, mice were sacrificed, and blood, urine and kidneys were harvested for further analysis. Blood glucose levels were measured from tail vein blood using a Bayer Contour glucose meter. Plasma cystatin C (R&D systems) and urine albumin (Bethyl laboratories) levels were determined by ELISA according to the manufacturer’s instructions. Urine creatinine levels were determined using an enzymatic mouse creatinine assay kit (CrystalChem), according to the manufacturer’s instructions.

Serum insulin (Merck, Darmstadt, Germany), urine albumin (Bethyl Laboratories, Montgomery, TX, U.S.), TGF-β1 (DAKEWE, Shenzhen, China) and NGAL (R&D Systems, Minneapolis, MN, U.S.) were detected by ELISA according to manufacturer’s instructions.

Fasting blood glucose was measured from tail blood by glucometer (Medisense, Abbott Laboratories, Bedford, MA). Urine was collected from mice housed in metabolic cages for 6 h. Heparinized whole blood was collected by cardiac puncture in anesthetized animals for analysis of HbA₁c and isolation of heparinized plasma. Urine creatinine levels were determined by the Jaffe rate reaction method. ELISA kits were used to assess levels of urine albumin (Bethyl Laboratories, Montgomery, TX) and plasma cystatin C (R & D Systems, Minneapolis, MN). Albuminuria was measured by calculating the urine albumin/creatinine ratio (ACR). HbA1c was measured by DCA Vantage Analyzer (Siemens, Camberley, UK).