A template search was

performed through the PDB database,

A template search was

performed through the PDB database, using the BLAST algorithm (Altschul et al., 1990) for the TsNP sequence, with the structure of lebetin 2 isoform alpha from Macrovipera lebetina (PDB code: 1Q01) selected. The following alignment properties check details with TsNP were found: E-value: 0.0181276; Score: 35.039 bits (79); Identities: 14/19 (74%); Positives: 17/19 (89%); Gaps: 0/19 (0%). The PDB 1Q01 structure was used as the template for the homology modeling. Multiple sequence alignments among the target (TsNP) and reference sequences were performed using the ClustalX program ( Thompson et al., 1997) with its default parameters. Adult male Wistar rats (weighing 260–320 g) click here from the Animal Facility of Universidade Federal do Ceará were used in the renal function experiments. The rats were kept in a housing room with controlled ambient humidity, room temperature maintained at 22 ± 2 °C, laminar air flux and 12 h light/dark

circles. All animal studies were performed according to Brazilian laws for animal experimentation and were approved by the Ethical Committee of Animal Experimentation of Universidade Federal do Ceará under the number 107/07. The rats (n = 6) were fasted for 24 h with free access to water before the experiment. The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). After careful dissection of the right kidney, the right renal artery was cannulated via the mesenteric artery without interruption of blood flow, as described by Bowman (1970) and modified by Fonteles et al. (1983). A modified Krebs-Henseleit Phosphoprotein phosphatase solution (MKHS, composition in mmol/L: 118.0 NaCl, 1.2 KCl, 1.18 KH2PO4, 1.18 MgSO4.7H2O, 2.50 CaCl2 and 25.0 NaHCO3) was used for the perfusion. Bovine serum albumin (BSA) fraction V (6 g) was added to 100 mL of MKHS, and this solution was dialyzed for 48 h at 4 °C against 10 volumes of MKHS. Immediately before the beginning of each perfusion

protocol, 100 mg of urea, 50 mg of inulin and 50 mg of glucose were added to the dialyzed solution (100 mL), and the pH was adjusted to 7.4. In each experiment 100 mL of MKHS were recirculated for 120 min. The perfusion pressure (PP) was measured at the tip of the stainless steel cannula in the renal artery. Samples of urine and perfusate were collected at 10 min intervals for the determination of sodium, chloride and potassium levels using ion-selective electrodes (Electrolyte Analyzer 9180, Roche™). Inulin levels were determined as described by Walser et al. (1955). Osmolality was measured with a vapor pressure osmometer (VAPRO® 5520,Wescor™). TsNP (0.1 μg/mL or 0.03 μg/mL) was added to the system 30 min after the beginning of each perfusion.

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