Mouse model of diabetic nephropathy

Animal studies were conducted in accordance with National Institute of Health Guidelines for the Care and Use of Laboratory Animals. All procedures were approved by the Ethics Committee of the University of Turin and the Italian Health Ministry (authorization number: 280/2016-PR). Eight-week-old male NSG mice were purchased from the animal facility at the Molecular Biotechnology Centre. Diabetes was induced via the intraperitoneal injection of STZ (37 mg/kg) that had been dissolved in freshly made 0.1 mol/l citrate buffer, at pH 4.5, for 4 consecutive days in order to avoid acute STZ toxicity, according to Animal Models of Diabetic Complications Consortium guidelines (available at http://www.amdcc.org)⁶¹.

Blood glucose levels were measured, after 4 hours of fasting, in blood from the tail-vein using a blood glucometer (GlucoMen LX Plus+, A. Menarini diagnostics, Florence, IT). The onset of diabetes was established by measuring glycaemia (up to 250 mg/ml) 10 days after STZ injection (T0). Glycaemia was monitored every 2 weeks and body weight and water up-take every week. After one month of diabetes, mice were randomly divided into the following groups: the HLSC EV, MSC EV and FIBRO EV groups, which were injected intravenously with 1 × 10¹⁰ particles each injection, once a week from day 30 (T30) for 4 weeks (5 injections) and the CTL group, which was injected with an equal volume of saline. Mice were sacrificed either 28 or 60 days post diabetes. At either day 60 (T60) or day 28 (T28), urine (12 hours collection in metabolic cage) and blood were collected for the evaluation of albuminuria, creatinuria, plasma creatinine and BUN. Kidneys were collected for histology and molecular analyses.