The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication NO.85-23, revised 1996), according to the animal welfare regulations of the Italian local authorities.
Male Sprague-Dawley rats weighing about 200 g were used (Charles River Italia, Lecco, Italy) and were allowed water and standard rat chow ad libitum. All the rats were maintained at 22 ± 1°C with a 12/12 hours light/dark cycle.
Ischemia/Reperfusion (Isc/R) model
The rats were anesthetized with an inhaled anesthesia mixture of halothane 2% (Hoechst, Milano, Italy) and oxygen. They were placed on a temperature-regulated table (38°C) (Ugo Basile, Comerio, Lecco, Italy) to maintain body temperature. Kidney ischemia (Isc) was induced by clamping the right renal artery and the right renal vein for 60 minutes with a microsurgical clamp. In the Isc/R group, at the end of the I period, the vascular clamp was removed and reperfusion of 60 minutes was performed. During the surgical procedure the heart rate and the mean arterial blood pressure (MABP) were monitored.
At the end of Isc or of Isc/R, blood samples were obtained by exanguination of rats at the aorta bifurcation level and kidneys were collected and processed for different studies. Blood and kidneys from EDTA-treated-not-ischemized rats were collected 90 minutes after EDTA administration (corresponding to 30 minutes EDTA pre-treatment+60 min Isc).
Measurement of mean arterial blood pressure
The right femoral artery was cannulated through a polyethylene catheter and connected to a pressure transducer for the measurement of MABP [15, 16]. The data was collected continuously by means of a computer and were calculated at baseline, at the end of EDTA pre-treatment (e.g 30 minutes after EDTA intravenous injection), at the end of Isc and at the end of postischemic R. In sham-operated rats the values were calculated 90 minutes after EDTA pre-treatment.
EDTA (calcium disodium EDTA) (Collalto, Brescia, Italy) used in human therapy was employed , and at the same dosage (e.g. 40 mg/kg body weight). The sterile drug solution of 2 g/10 ml was opportunely diluted in physiological saline and administered by left intrafemoral vein slow infusion.
The inhibitor of NO synthases L-NAME [N(omega)-nitro-L-arginine methyl ester], when required, was injected simultaneously with the EDTA through the intrafemoral vein at the dose of 30 mg/kg body weight, 30 minutes before the induction of Isc or Isc/R.
The rats were randomly allocated to 4 study groups, each composed of 15 rats: group 1, controls; group 2, sham operated: the rats underwent the same surgical procedure, except that the clamp was not applied; group 3, Isc: ischemia was induced for 60 min; group 4, Isc/R: ischemia was induced for 60 min, followed by 60 min reperfusion at room temperature. Other identical 4 groups were studied, in which EDTA treatment was performed. In groups 3 and 4 intrafemoral injection of physiological saline 30 minutes before clamping was performed. The 3 and 4 EDTA-treated groups received a single intravenous injection of EDTA 30 minutes before clamping. In groups 1 and 2 intrafemoral injection of physiological saline or EDTA was performed 90 minutes before kidney removal (= 30 minutes EDTA pre-treatment+60 min Isc).
To take in consideration that EDTA could lead to increase in NO plasmatic levels through increase in eNOS expression, we further performed histological evaluations on two additional groups of rats, to verify whether the eNOS inhibitor L-NAME was able to block the protective effect of EDTA in renal ischemic injury. In such groups the animals were simultaneously treated with EDTA and L-NAME 30 minutes before the induction of Isc (group 5) and 30 minutes before the induction of Isc/R (group 6).
Serum creatinine was measured using a modified Jaffe's reaction, and blood urea nitrogen was measured on the AEROSET system (Abbott Laboratories, Abbott Park, IL) .
Histopathology and immunofluorescence microscopy
Kidneys were excised, decapsulated, dissected into 4 pieces along the major ax, fixed by immersion in 4% paraformaldehyde in Dulbecco's PBS (DPBS) overnight at 4°C, cryo-protected in 10% sucrose in DPBS, then embedded in Tissue-Tek medium and frozen in liquid nitrogen. Cryostat-cut four sections/animal (5 μm thick) were submitted to Hematoxylin/Eosin stain; renal damage was evaluated as tubular epithelial cell necrosis, tubular dilation, protein casts and medullary congestion (18). The alterations were semi-quantitatively graded by a pathologist blind to the nature of the experiments. The grading was performed by the following criteria: - =absent, + = barely present, ++ = moderate, +++ = severe. Expression of eNOS, e.g. the endothelial form of the constitutive NO synthase, was assessed on serial sections, with the use of a specific monoclonal antibody (BD Pharmingen, Franklin Lakes, NJ), followed by a Rabbit-anti-Mouse IgG- AlexaFluor488 (Molecular Probes, Eugene, OR). Observations were performed by using an Eclipse 55i microscope (Nikon, Tokyo, Japan), digital images acquired with DS-L1 camera and LUCIA G software (all from Nikon) and mounted using AdobePhotoshop CS software.
The expression of Mac-1 was evaluated by following FACS analysis. Whole blood was incubated with 0.5 μg of FITC-conjugated CD11b monoclonal antibody (clone WT5, isotype mouse IgA, K) (Pharmingen, San Diego, CA) for 20 minutes in ice. After erythrocyte lysis, samples were run on a FACscan (Becton-Dickinson, Mountain View, CA) and gated on PMN parameters. Results are expressed as arbitrary units of mean fluorescence intensity (MFI, a.u.).
Nitrite/Nitrate (NO2-/NO3-) determination
The rats were bled off at the aorta bifurcation level. Blood was collected in the presence of 0.065 mM citric acid (Riedel, Hannover, Germany), 0.085 mM sodium citrate (Farmitalia, Milan, Italy) and 2% glucose monohydrate (Riedel) in the blood: anticoagulant ratio of: 7:1. Samples were obtained from rats immediately after the end of each treatment or surgical procedure.
NO release was determined spectrophotometrically  by measuring the nitrate/nitrite (NO2
-) concentration in plasma samples from arterial non coagulated blood. Briefly, whole blood was centrifuged and plasma samples were collected, incubated for 30 min at 37°C in the presence of 0.2 U/ml Aspergillus nitrate reductase (Boehringer-Mannheim, Milan, Italy), 50 mM HEPES buffer (pH 7.4), 5 μM flavin adenine dinucleotide (Sigma Aldrich), and 0.1 mM NADPH (Sigma Aldrich). Then, lactate dehydrogenase (Boehringer Mannheim) and sodium pyruvate (Sigma Aldrich) were added to a final concentration of 10 U/ml and 10 mM, respectively, and the samples were incubated for 10 minutes at 37°. The Griess reagent (Sigma Aldrich) was added to the samples (100 μl), and absorbance was measured at 540 nm after 15 minutes incubation at room temperature. Standard curves with increasing concentrations of sodium nitrate and sodium nitrite were run in parallel.
In vivo permeability assay
The assay was performed as described . Briefly, the exit of albumin from vessels into the parenchyma of rat kidneys was assayed. The dye solution contained 0.4% albumin (Sigma Aldrich) and 0.5% trypan blue (Sigma Aldrich) in saline. Following laparatomy, animals were perfused with 5 ml dye-solution through the right renal artery for 10 minutes. The perfusate was drawn from the right renal vein. The right kidney was washed with saline in vivo, removed, weighted, suspended and homogenized in buffered phosphate solution at pH 7.4 (1 g tissue dissolved in 3 ml buffer). In treated animals, after halothane anesthesia, EDTA (40 mg/kg) was injected intravenously (through the femoral vein), followed by rat TNFα (R&D System, Abingdon, UK) (0.1 ng/g). TNFα and EDTA, alone or together, were injected 30 minutes before kidney dye perfusion.
Tissue extracts were centrifuged, the supernatants recovered and treated with 10% deoxycholic acid (sodium salt monohydrate, Sigma Aldrich) in saline, to remove lipid interference. Dye was evaluated by spectrophotometer analysis (Pye Unicam SP6-550, Cambridge, United Kingdom) at 540 nm.
The results are expressed as the mean ± SEM of 15 animals in each group. They were analyzed using a two way analysis of variance followed by Bonferroni t-test. The results were considered statistically significant when p < 0.05.