The study protocol was approved by the locally appointed Ethics Committee. The mice were housed and cared for according to the Principles of Laboratory Animal Care protocols (NIH publication No. 85-23, revised 1985) and national laws and regulations and had free access to water and food. Six-week-old adult female Apoe-null mice (Charles River, Calco, Italy) were rendered diabetic by streptozotocin (Sigma, St Louis, MO, USA) administered by intraperitoneal injection at a dose of 50 mg/kg body weight for 5 consecutive days. Mice injected with citrate buffer served as non-diabetic controls (Cont). Diabetic mice were divided into four groups, each consisting of ten mice: untreated (Diab); mice treated with 60 mg/kg body weight DCO (Flamma, Chignolo d’Isola, Italy) dissolved in the drinking water for the entire duration of the study (20 weeks, DCO-Extended); mice treated with DCO for 11 weeks only, either starting immediately after diabetes induction and continuing up to week 11 (DCO-Early) or started at week 9 and continuing to week 19 (DCO-Late) followed by 1 week of wash-out to avoid potential interference of DCO plasma levels on the comparison with DCO-Early.
At the end of the 20 week period, the mice were placed into metabolism cages to collect urine. The next day, mice were anaesthetised by intraperitoneal injection of ketamine (Imalgene, 60 mg/kg, Merial, Milan, Italy) and xylazine (Rompum, 7.5 mg/kg, Bayer, Milan, Italy) and the heart (with attached aorta) and both kidneys were sampled and weighed. The aortic sinus, the brachiocephalic artery (BCA) and the right kidney were fixed in 4% (wt/vol.) formaldehyde. Then, the aortic sinus was embedded in Optimal Cutting Temperature embedding medium (Tissue-Tek, Torrance, CA, USA) for frozen section preparation and the BCA and the right kidney specimens were embedded in paraffin. The abdominal aorta was opened longitudinally, layered on ovalbumin-coated glass slides, allowed to dry for 1 h and then fixed in formalin for 24 h for en face preparation . The left kidney and the thoracic part of the descending aorta were frozen in liquid nitrogen for subsequent mRNA and protein extraction. In selected mice, termination was preceded by measurement of BP by the tail-cuff method, using the CODA System (Kent Scientific, Torrington, CT, USA).
Blood and urine measurements
Blood glucose was measured using an automated colorimetric instrument (Glucocard G meter; Menarini, Florence, Italy), and serum cholesterol and triacylglycerols by enzymatic colorimetric methods (Roche Diagnostics, Milan, Italy). Serum levels of isoprostane 8-epi-prostaglandin (PG) F2α were assessed using an ELISA kit (Cayman, Ann Arbor, MI, USA) and those of AGEs were measured by a competitive ELISA technique [17, 19, 20]. Serum and urine creatinine levels were measured by HPLC and proteinuria and albuminuria were assessed using the Bradford dye-binding protein assay kit (Pierce, Rockford, IL, USA) and the Mouse Albumin ELISA kit (Bethyl, Montgomery, TX, USA), respectively, and results were normalised to urine creatinine concentration [17, 20].
For the assessment of atherosclerotic lesions [17, 19], the extent of lipid accumulation was evaluated in en face preparations after staining with Oil Red O. Lesion area was quantified at the aortic sinus and BCA level after staining with Oil Red O and the Weigert–van Gieson method for elastic and collagen fibres , respectively. Five cryosections of aortic sinus (10 μm thick, every other section) and four sections (5 μm thick) at 200, 400, 600 and 800 μm from the branching point of the BCA were evaluated. The necrotic core area and collagen content were measured in five sections of the aortic sinus (50 μm apart) and in four sections of the BCA (200 μm apart) stained with the Weigert–van Gieson method. For the assessment of renal lesions , 4 μm sections were stained with periodic acid–Schiff (PAS) to allow glomerular and tubule-interstitial damage to be examined by light microscopy. Moreover, the areas of at least 60 glomerular tuft profiles per sample were measured and the harmonic mean of the profile area (mean glomerular area, mGA) was obtained. Then, PAS-positive material was quantified and expressed as a percentage of the glomerular tuft area (fractional mesangial area, fMA), and the mean mesangial area (mMA) was calculated by the formula: (fMA × mGA)/100. All measurements were performed with the aid of the Optimas 6.5 image analysis system (Bioscan, Washington, DC, USA).
Immunohistochemical analysis [17, 19, 20] was performed to assess the aortic and kidney content and distribution of markers of murine macrophage activation (F4/80), oxidative stress (nitrotyrosine) and carbonyl stress (N
ε-carboxymethyllysine, CML). Nitrotyrosine was chosen as a marker of free radical damage since its production does not depend on RCS levels and, hence, is not directly influenced by DCO treatment, at variance with CML and the adducts of malonylaldehyde or 4-hydroxy-2-nonenal. The expression of active caspase-3, a marker of apoptotic cells, α-smooth muscle actin (α-SMA), a marker of vascular smooth muscle cells (VSMCs) and receptor for AGEs (RAGE) in the aorta, and of collagen IV in the kidney, were also assessed (see electronic supplementary material [ESM] Table 1 for primary antibodies). Sections were analysed using the Optimas 6.5 image analysis system.
Quantitative real-time PCR
Total RNA was extracted from aorta and kidneys by the guanidine thiocyanate–phenol–chloroform method using TRIzol Reagent (Invitrogen Italia, San Giuliano Milanese, Italy). Quantitative real-time PCR (qRT-PCR) was used for assessing the gene expression level of multiple disease markers . The transcripts for the following proteins were measured in the aorta: the inflammatory markers F4/80, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, the antioxidant enzymes CuZn-superoxide dismutase (CuZn-SOD), catalase and glutathione peroxidase 1 (GPX1) and hypoxia inducible factor-1α (HIF-1α), the transcriptional activity of which is increased by AGEs  and inhibited by carnosine . The transcripts for the following were measured in the kidney: fibronectin, collagen IV, TGF-β, MCP-1, CuZn-SOD, catalase, GPX1 and HIF-1α.
Results are expressed as means ± SD. Statistical significance was evaluated by one-way ANOVA followed by the Student–Newman–Keuls test for multiple comparisons. A p value <0.05 was considered significant. All statistical tests were performed on raw data.