Urinary protein electrophoresis was performed on agarose gel followed by violet acid on Hydrasys (SEBIA, Lisses, France); each sample was run in duplicate. Albumin concentrations were derived after scan densitometry of the gel by proportion with urinary total proteins. In order to evaluate the different origin of urinary proteins (glomerular or tubular), urine samples were analyzed by agarose/sodium dodecyl sulfate (SDS) gel electrophoresis painted with violet acid.Morphological assessment of damageAnimals were euthanized with an overdose of pentobarbital (200 mg/kg, i.p.) at baseline and 3 and 7 hours after CLP or sham-operation and processed in different ways according to the histological technique used for morphological analysis.Structural and ultrastructural analysis Kidney samples were fixed in a solution containing 2% glutaraldehyde, 2% sucrose, 0.1 mol/L sodium cacodylate phosphate and 2% lanthanum nitrate for 4 hours at room temperature, and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, for 1 hour at 4��C for light microscopy and transmission electron microscopy. Then the specimens were dehydrated in graded acetone, passed through propylene-oxide and embedded in Epon 812. Semi-thin sections, 2 ��m thick, were stained with toluidine blue-sodium tetraborate and observed under light microscopy. For quantitative analysis, the number of damaged renal corpuscles was evaluated by counting 10 random 600625 ��m2 optical square fields (40 �� ocular) under an inverted phase-contrast Nikon DIAPHOT 300 microscope (NIKON, Melville, NY, USA) in each experiment. The number of damaged corpuscles counted by two different observers was expressed as the percentage of the total renal corpuscles. Ultrathin sections were stained with uranyl acetate and alkaline bismuth subnitrate and then examined under a transmission electron miscroscoey (Jeol 1010, Tokyo, Japan) at 80 kV.Confocal immunofluorescence After the pentobarbital overdose, a midline incision was made in the abdomen and thorax of 16 rats; the kidneys were immediately fixed by transcardial perfusion by flushing with phosphate-buffered saline (PBS) for 1 minute followed by 4% paraformaldehyde in PBS buffer for 3 minutes. All solutions were maintained at pH 7.4 at 4��C. Kidneys were removed and post-fixed in 4% paraformaldehyde in PBS overnight, then transferred to PBS containing 30% sucrose and finally frozen at -80��C. For immuno-staining, cryostat sections, 10 ��m thick, were permeabilized with cold acetone for 10 minutes, blocked with a solution containing 0.5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and 0.