2.1. Animal model

Adult male Sprague-Dawley rats weighing 150–250 g, were obtained from the animal facility at the Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Argentina.

The experimental protocols and euthanasia procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Facultad de Medicina, Universidad de Buenos Aires, Argentina (CICUAL; CD Resolution No. 2356/11). All the procedures were performed in accordance with the EEC guidelines for care and use of experimental animals (EEC Council 86/609).

Rats were randomly divided into four groups (n = 6 per group). Groups 1 and 2 (experimental groups) were injected intraperitoneally with Streptozotocin (STZ) (Sigma-Aldrich Corp., San Luis, MO, USA) to induce diabetes. Rats were fasted overnight and injected with 65 mg/kg of STZ diluted in freshly prepared citrate buffer (pH 4.5). Induction of diabetes was confirmed 72 h post STZ injection by measuring glycemia in tail vein (Freestyle optium™, Abbot, Oxon, UK). Animals presenting glycemia >350 mg/dL were included in the study. Groups 3 and 4 (control groups) were injected with PBS by the same route. Groups 1 and 3 were studied at 15 days and Groups 2 and 4 at 5 months. The rats were individually housed under controlled conditions of illumination, humidity, and temperature, with food and water being available ad libitum. 24 h prior to sacrifice, to study the renal function animals were placed on metabolic cages for urine collection and blood samples were obtained by cardiac punction. Serum and urinary creatinine levels and urinary protein concentration were determined using a Cobas C311 Autoanalyzer (Roche Diagnostics, Basel, Switzerland). Creatinine clearance was also calculated by the following formula:

Urinary osmolality was also measured using a pressure vapor osmometer VAPRO™ (Wescor Inc., Logan, UT, USA).

Finally, rats were anesthetized (100 μg ketamine and 10 μg diazepam per g of body weight, intraperitoneally). Left kidney was removed and conserved at -20 °C to molecular studies and then the animals were perfused with 4% paraformaldehyde. Right kidney was removed, and the tissues were fixed in 10% neutral formalin.

Histological PAS sections of each groups of rat kidneys were also analyzed.