Does prolonged pneumoperitoneum affect the kidney ?

ARTICLE INFO _________________________________________________________ ___________________ Vol. 39 (1): 30-36, January February, 2013 doi: 10.1590/S1677-5538.IBJU.2013.01.05 IBJU | DOES PROLONGED PNEUMOPERITONEUM AFFECT THE KIDNEY? 31 To address this issue, in the present study we induced in rats a prolonged 8 mmHg Pp and then investigated the short-term oxidative stress alterations and the long term morphological changes in the kidney applying stereological and electron microscopic methods. MATERIALS AND METHODS Animals Twenty-eight Wistar male rats aged 4 to 6 months and weighing 250 to 360 g were used in this study. The rats were kept in a room with controlled temperature (25 ± 1 oC) and with an artificial dark-light cycle (lights on from 7:00 am to 7:00 pm). They were fed standard rat food and water ad libitum. All experiments were performed in accordance with the Brazilian laws for scientific use of animals, and the project was approved by the local ethical committee. Groups The rats were randomly assigned into a Sham group (n = 14), which was submitted only to anesthesia for 210 minutes, and a Pp group (n = 14), which underwent, under anesthesia, 180 minutes of Pp at 8 mmHg followed by 30 minutes of deflation. Immediately after these procedures, 7 animals were killed in each group and their kidneys were used for oxidative stress analyses. The remaining 7 rats from each group were killed 6 weeks later and their kidneys were used for stereological and electron microscopy analyses. All analyses were blindly performed. Pneumoperitoneum The animals were anesthetized by intraperitoneal injection of ketamine (80 mg/kg body weight) and xylazine (10 mg/kg body weight) with spontaneous breathing during the experiment. In the Pp group, a 21-gauge needle was inserted into the abdominal cavity and a Pp at 8 mmHg was established with CO2 by using an electronic insufflator. After 180 minutes of Pp, the abdominal cavity was deflated, and the animals remained anesthetized for 30 minutes. Stereological analyses The volume of the right kidneys was estimated by the Sherle ́s method (8); then the kidneys were sectioned frontally, fixed in 10% formaldehyde and routinely processed for paraffin embedding. Serial 5 μm sections of the entire kidney were obtained and stained with hematoxylin and eosin (HE). The cortical-medullar ratio was estimated by using the Cavalieri principle (9) and the absolute cortical volume (CV) was calculated by multiplying the cortical-medullary ratio by the renal volume. The left kidneys were sectioned in small fragments, fixed in 10% formaldehyde and routinely processed for obtaining 5 μm thickness HE stained slices. From each animal, different randomly sections of the renal cortex were obtained, from which 26 histological fields were captured at a 200x magnification and analyzed. A M42 test-system was applied to obtain the glomerular volume density (Vv[glom]) by the point-counting technique (9). The volume weighted mean glomerular volume (VWGV) was estimated by using the point-sampled intercepts method (10,11) analyzing 50 glomeruli per animal. The estimation of the total number of glomeruli per kidney was calculated by multiplying the CV by the Vv [glom] and dividing the result by the VWGV (12). Electron microscopy analyses Small fragments of the left kidneys from the Sham and Pp groups were used for investigating podocyte effacement. Samples were processed for scanning and transmission electron microscopy as previously described (13). Briefly, the tissue was minced and fixed in 2.5% glutaraldehyde overnight and post-fixed in 1% OsO4. For scanning electron microscopy, the fragments were dehydrated in ethanol, critical point-dried with CO2 and sputter-coated with gold-palladium and examined on a JEOL5800 scanning electron microscope. For transmission electron microscopy, the samples were gradually dehydrated in acetone and embedded in Epon. Ultra-thin sections were stained in 5% uranyl acetate and 1% lead citrate and then observed on a JEOL1210 transmission electron microscope. Podocyte effacement was assessed qualitatively by scanning and transmission electron microscopy, and quantitatively by transmission electron microscopy (14). The quantification was IBJU | DOES PROLONGED PNEUMOPERITONEUM AFFECT THE KIDNEY? 32 carried out by measuring the length of foot processes in contact with the basement membrane using the software ImageJ. At least 100 foot processes per animal were used to calculate individual mean. Oxidative stress analyses One fragment of the left kidney was washed with ice-cold 0.1 M phosphate buffer, pH 7.4 and stored at -20 oC. Then it was homogenized in the same buffer at a concentration of 0.1 (g tissue/ mL), centrifuged at 10000g at 4 oC for 15 min. Supernatants were removed and stored at –20oC until analyses. Malondialdehyde (MDA), which is formed by lipid peroxidation, was assayed by a colorimetric method using thiobarbituric acid (15). Protein oxidation was assessed by measuring the protein carbonyl content after reacting with dinitrophenylhydrazine (16). Protein carbonyl (nMol/mg) and MDA (nMol/mg) contents were expressed in relation to tissue protein concentration, which was measured with the Lowry method (17). Statistical analysis Student’s-t-test was used for all mean comparisons. For all comparisons p < 0.05 was considered significant. Analyses were performed using GraphPad Prism software.