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Prevention of renal injury after induction of ozone tolerance in rats submitted to warm ischemia

Título
Prevention of renal injury after induction of ozone tolerance in rats submitted to warm ischemia
Autores
  1. Barber, 1 S. Menéndez,2 O. S. León,3
  2. O. Barber,1 N. Merino,3, J. L. Calunga,2 E. Cruz1 y V. Bocci4,CA

1Instituto de Ciencias Básicas y Preclínicas ‘ Victoria de Girón’, Havana, 2 Centro de Investigación de Ozono, P.O. Box 6880, Havana; 3 Centro Nacional de Investigación Científica, Havana, Cuba; 4 Instituto General de Psicología, Universidad de Siena, Vía Laterina 8, 53100 Italia.

CA Autor Correspondiente

Tel. (+39) 577 263856

Fax: (+39) 577 263861 Email: fisgen@unisi.it

Revista o sitio electrónico (URL) donde se publicó

Mediators of Inflammation, 8, 37 -47 (1999)

Fecha de Recibido

1998-11-02

Fecha de Publicación

1998-12-18

Tissue ischaemia followed by reperfusion with oxygenated blood occurs in a number of clinical situations. In order to improve the success rate of renal transplantation, the timing of warm and cold ischemia is a major determinant for the kidney’s viability. 1,2

After reimplantation, owing to reperfusion with the oxygenated blood, the ischaemic kidney may develop tubular necrosis and the recipient needs to undergo dialysis. Furthermore, in order to avoid rejection, treatment with immunosuppressor drugs may, on one side worsen the renal damage, or become overtly toxic.1 Thus any prophylactic approach aiming at preserving the kidneys is of a crucial importance.

Recent advances in understanding the fundamental mechanisms of post-ischaemic injury have suggested that tissue injury is associated with higher oxygen (O2) tension in the tissue at the time of reperfusion. 2,3

The reoxygenation leads to a massive production of reactive oxygen species (ROS), generated through several cytoplasmatic or mitochondrial mechanisms, inducing an unbalance between oxidants and antioxidants, i.e. an oxidant stress, which contributes to tissue injury.2,4-6 ROS among which superoxide anion (O2-.) hydrogen peroxide (H2O2), hydroxyl radical (OH.), not only can damage cells by oxidizing nucleic acids, proteins and polyunsaturated lipids,7 but many ultimately led to cell death. There is no doubt that ROS, if unquenched, can compromise renal function by impairing glomerular filtration and tubular reabsorption. 8

In normal conditions, cells contain a powerful and articulate endogenous defense against ROS9 such as antioxidant enzymes, namely superoxide dismutases (SOD), catalase, glutathione peroxidase, thioredoxin reductase, or nonenzymatic components such as aerobic and uric acid, reduced glutathione, βcarotene, lycopene, vitamin E, bilirubin, etc. 2,8,9 In several pathologic situations and after ischaemia, these defense mechanisms can be overwhelmed allowing the ROS to exert their deleterious effect.2

Taking into account that ischemia-reperfusion is a process largely mediated by ROS generation2-6 and that a prolong and judicious administration of O3 is able to stimulate the endogenous antioxidant systems, 11-14 and thereby to oppose the oxidative stress, we thought it worthwhile assessing renal morphology and function and a few biochemical parameters in rats undergoing a controlled, warm renal ischaemia.

Materials and methods

Animals and sample preparation

Forty adult male Wistar rats (250-260g) were maintained in an air filtered and temperature conditioned room (20-22 0C) with a relative humidity of 50-52%.

Rats were fed with a standard commercial diet and water ad libitum. O3 was generated by OZOMED equipment (Ozone Research Center, Cuba) from medical grade oxygen by means of a silent electric discharge, representing about 3% of the gas mixture (O3+O2). Rats received 15 ozone treatments, by rectal insufflation, performed with a suitable polythene cannula connected to a syringe, once daily, 2.5 -2.6 ml with O3 concentration of 50 µg/ml (representing a dose of 0.5 mg/kg weight), before the renal ischaemia reperfusion damage. This schedule and O3 dosing has proved to be optimal in a previous study.13

Under constant ischaemia of 30 min, we allowed a reperfusion period of exactly 3 h. Heparin (50 U) was administered via the subcutaneous route. Immediately thereafter, within the following 10min. we assessed the renal plasma flow (RPF) and the glomerular filtration rate (GFR) by means of plasma clearance of p-amino-hippurate (PAH) and inulin, respectively. A constant plasma concentration of both substances was used (2mg of PAH and 20 mg of inulin in 100ml of saline solution) by a continuous perfusion through the left femoral vein at a rate of 0.15 ml/min., after a loading dose of 0.8 ml of PAH (12 mg/ml) and 0.8 of inulin (2mg/ml). For these analyses, blood was withdrawn by intracardiac puncture and urine were collected from the bladder. Thereafter the rats were euthanized under deep anesthesia. Representative samples of different kidneys portions were taken for histophatiological studies and tissue homogenates. Kidney homogenates were obtained using a tissue homogenator Edmund Bulher LBMA at 4 0C. The homogenates were prepared in 50 mM KCl/histidine buffer pH 7.4, 1:10 (W/V) and were spun down with a sigma Centrifuge 2K15, at 40 C and 8500 X g for 20 min. The supernatants were used for biochemical determinations.

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