Ozone Biological Response in Kidneys of Rats Submitted to Warm lschemia

Author Jose Luis Calunga 1
Author Ernesto Barber2
Author Silvia Menendez* 1
Author Nelson Merino3
Author Eduardo Cruz3
Publication 10zone Research Center. POBox 6880, Havana, Cuba
Publication 2Institute of Basic and Preclinical Sciences “Victoria de Giron”, Havana, Cuba
Publication 3Center for Research and Biological Evaluation, Havana University, Institute of Pharmacy and Food Sciences, POBox 6079, Havana, Cuba
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Ozone Biological Response in Kidneys of Rats Submitted to Warm lschemia


A biochemical and morphofunctional renal study, applying different sessions of rectal ozone before a warm ischemia, was performed. Rats were divided in:!­ control, a medial abdominal incision was performed for the exposure of the kidneys; 2-ischemia, animals with a bilateral renal ischemia (30 min), with subsequent reperfusion (3 h); groups 3, 4 and 5-ozone + ischemia, as group 2, but with previously treatment of 5, I 0 and 15 sessions of rectal ozone, respectively; groups 6, 7 and 8-oxygen + ischemia, as groups 3, 4 and 5, respectively, but using rectal oxygen. A significant decrease of the flow and renal filtration, with high values of fructasamine and phospholipase A, in the ischemia and oxygen groups, with respect to control and ozone groups was obtained. Control and  ozone groups, without any statistical difference among them. Morphological alterations were significantly  less in the groups pretreated with ozone, with better results for I 0 and 15 sessions.


It have been demonstrated that reactive oxygen species (ROS) play a key intermediary role in the pathophysiologic processes of clinical and experimental renal diseases (1,2). They can promote DNA scission and base modification, inactivate plasma proteins, crosslink membrane proteins and induce lipid peroxidation in the polyunsaturated fatty acids of membrane lipids (3).

Tissue injury following renal ischemia represents the injurious effects of ROS and anoxia/hypoxia in varying degrees.(4) The major damage. is apparently  secondary  to reperfusion or reoxygenation. This reoxigenation leads to a massive production of toxic free radical species generated through several cytoplasmatic or mitochondrial  mechanisms, producing an oxidative stress, which contributes to tissue injury. (3-7). Tissue ischemia, followed by reperfusion with oxygenated blood, occurs in a number of clinical situations,  as

surgical revascularisation of the renal artery and after kidney transplantation. In order to improve the success rate of renal transplantation, the timing of warm and cold ischemia is determinant for kidney’s viability (8) After reimplantation, the kidney may develop an acute tubular necrosis, and the recipient must required to undergo dialysis. Any prophylactic approach aiming at preserving the kidney is of great clinical importance.

In normal conditions, cells contain endogenous defense systems, as superoxide  dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) or non enzymatic components such as glutathione (GSH) and vitamins E, A, C (6). The SOD is the enzyme which  detoxify  the primary product of ROS generation, the superoxide anion radical (formed by the action of xanthine oxidase), leading to the production of hydrogen peroxide. This ROS  is  then converted to water, through the action of glutathione peroxidase or directly  to  water  and oxygen by means of catalase (9). In several pathologic situations, an unbalance  between oxidants and antioxidants occurs, and  these  defense  mechanism  can  be  overwhelmed, allowing the ROS to exert their deleterious effect (6).

Taking into account that ozone (03), by means of an oxidative preconditioning mechanism, is able to afford protection against cellular damage mediated by free radicals, stimulating the cellular antioxidant enzymes (8, 10-14), we decided to study, in rats, the influence of different sessions of rectal ozone before a warm ischemia, in the renal morphology and function, and in some biochemical parameters.

Materials and Methods


Eighty adult male Wistar rats (250-260 g) were maintained in an air filtered and temperature conditioned room (20-22 °C) with a relative humidity of 50-52 %. Rats were fed with standard laboratory chow and water ad libitum and were kept under an artificial light/dark cycle of 12 h.

Treatment Schedule and Renal Ischemia

03 was generated by an OZOMED equipment (Ozone Research Center, Cuba), from medical grade oxygen by means of a silent electric discharge, representing about 3 % of the gas mixture (03+02). The ozone concentration was measured by using an UV spectrophotometer at 254 nm. The ozone dose is the product of the ozone concentration, expressed as mg/L, by the gas (02 + 03) volume (L). By knowing the body weight of the rat, the ozone dose is calculated as 1 mg/kg, as in our previous papers (8, 10-13).

Animals were allocated randomly to 8 experimental groups, of 10 animals each: I -control, animals  were  anesthetized,  using  sodium  pentobarbital   at  doses  of  30  mg/Kg  of  weight,

rece1vmg 50 I.U. of heparin by intraperitoneal injection. Afterwards, a laparotomy was performed for the sham exposure of the kidneys with successive laparorrhaphy; 2- positive control group (ischemia): animals were processed in the same way as group 1, but after the kidney exposition they were submitted to a bilateral renal ischemia. Both renal arteries were cross-clamped for 30 min, with subsequent reperfusion during 3 h, before the morphological, functional and biochemical renal study; groups 3, 4 and 5-ozone groups (02 + 03 and ischemia), as group 2, but the animals were previously treated with 5, 10 and 15 sessions of a gas mixture composed of 02 + 03 (2.5-2.6 ml with 03  concentration of 50 µg/ml, representing a dose of 0.5 mg/kg weight), by rectal insufflation, once per day, respectively; groups 6, 7 and 8-oxygen groups (02 + ischemia), as groups 3, 4 and 5, respectively,  but using rectal oxygen

(13 mg/kg weight) instead of the gas mixture composed of 02 + 03.

Sample Preparation

Under constant sodium pentobarbital  anesthesia, after a renal ischemia of 30 min, we allowed a reperfusion of 3 h. Heparin (50 UI) was administered by  intraperitoneal injection. Immediately, within the following 10 min were collected urine samples in the bladder for the renal function determinations: 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 (2 mg of PAH and 20 mg of inulin in 100 ml of saline solution) by a continuos 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  ml  of inulin  (2 mg/ml). Blood was withdrawn by intracardiac puncture (2 ml of blood were extracted). Thereafter the animals were euthanized by ether anesthesia.

Representative samples of different kidney portions were taken for histopathological studies and tissue homogenates. Kidney homogenates were obtained using a tissue homogenator Edmund Bulher LBMA at 4 °C. The homogenates were prepared in 50 mM KCI/histidine buffer pH 7.4, 1:10 (w/v) and were spun down with a Sigma Centrifuge 2K15, at 4 °C and 8500 g for 20 min. The supernatants were taken for biochemical  determinations.

Biochemical  determinations

PAH and inulin were determined in deproteinated plasma and urine samples by  cadmium sulfate (15), using for PAH a photocolorimetric technique as modified by Smith and Tinkelstein (16). Inulin was measured by the  direct method of resorcinol without alkaline treatment  (17).

Kidney homogenates were assayed for total SOD (Cu/Zn and Mn SODs) activity, determining the capacity of the enzyme of inhibiting the autoxidation of pirogallol by 50 % (18). The phospholipase A activity was determined according to a standard procedure ( 19) and fiuctosamine by means of a colorimetric procedure (20). The proteins were measured by a standard Coomassie Blue method (21). Ultraspect Plus  Spectrophotometer  from  Pharmacia LKB was used for spectrophotometric methods.

Histological study

Samples of rat kidneys were taken and fixed in 10 % neutral buffered formalin, processed and embedded in paraffin. The histological sections, stained with hematoxylin and eosin, were examined by a pathologist unaware of the treatment schedule.

Statistical analysis

The statistical analysis was started by using the OUTLIERS preliminary tests for detection of error values. Afterward the Anova method (one way analysis of variance) was used followed by homogeneity variance test (Bartlett-Box). In addition, a multiple comparison test was used (Duncan test) and  for the comparison of two groups, the Student’s t test was done. For the analysis of the biochemical parameters Mann-Whitney test was applied. Results are presented as mean ± standard deviation (SD). Different letters indicate a statistical  significance  of at least p <  0.05.


Table I shows the renal flow measured by means of the clearance of PAH and the GFR measured by means of plasma clearance of inuline. A significant decrease of PAH and Inuline clearances  were  observed  in  groups  2  (ischemia)  and  3,  4  and  5  (02-5,  10 and  15 d  +

ischemia, respectively)  in comparison with either groups  1 (control) or 6, 7 and 8 (03-5,  10

and 15 d + ischemia, respectively). Among either groups 2, 3, 4 and 5 or groups 1, 6, 7 and 8 there were no statistically significant differences.

Table I. Plasmatic clearance of p-amino-hippurate  (PAH) and Inulin in the different experimental groups


Experimental Groups (n=lO)



Inuline mVmin/1002
I -Control 3.08 ± 0.73a 0.60 ± 0.20a
2-Ischemia 1.66 ± 0.82b 0.32 ± 0.26b
3-02(5d)+ischemia 1.58 ± 0.70b 0.33 ± 0.27b
4-02(1Od)+ischemia 1.49 ± 0.67b 0.35 ± 0.23b
5-02(15d)+ischemia 1.16 ± 0.61b 0.43 ± 0.20b
6-03(5d)+ischemia 2.45 ± 0.81a 0.49 ± 0.15a
7-03(1Od)+ischemia 2.66 ± 1.06a 0.53 ± 0.18a
8-03( 15d)+ischemia 3.51 ± 0.90a 0.72 ± 0.35a


Values represent mean ± SD. Statistical significance between a and b of at least p<O. 05

Table II shows the biochemical parameters measured in kidney homogenates. The phospholipase A activity (PLA2) in the groups 6, 7 and 8 (03-5, 10 and 15 d + ischemia, respectively) did not differ from the control group and values were significantly lower in comparison with the group 2 (ischemia) and groups 3, 4 and 5 (02-5, 10 and 15 d + ischemia, respectively). No difference was observed among the groups 2, 3, 4 and 5. A similar trend was observed in regard to the fructosamine concentration, that yielded significantly  lower values in control and 03-5,  10 and  15 d + ischemia groups in comparison with the ischemia

and Oi-5, 10 and  15 d + ischemia groups. No statistical differences among groups 1, 6, 7 and

8 and among groups 2, 3, 4 and 5 were observed. As was expected, the renal SOD activity showed a significant increase in the 03-5, 10 and 15 d + ischemia groups in comparison with control, ischemia and 02-5, 10 and 15 d + ischemia groups, No statistical differences among groups 6, 7 and 8 and among groups 1, 2, 3, 4 and 5 were observed.

Table II. Results of phospholipase A2 (PLA2), fructosamine and superoxide dismutase (SOD) in the different experimental groups.

Experimental Groups (n=lO)




L\ D.O


U/mg proteins

I-Control 22.43 ± 12.15a 0.013 ± O.O lOa 742 ± 182a
2-Ischemia 104.74 ± 33.77b 0.020 ± 0.005b 964.48 ± 184.00a
3-02(5d)+ischemia 109.40 ± 24.60b 0.016 ± 0.001b 856.99 ± 103.80a
4-02( I Od)+ischemia 100.91 ± 23.12b 0.017 ± 0.001b 877.73 ± 103.2la
5-02( l 5d)+ischemia 92.54 ± 22.6l b 0.019 ± 0.001b 996.12 ± i28_6r
6-03(5d)+ischemia 47.71 ± 5.70a 0.014 ± 0.002a 1032.0 ± 99.6b
7-03(I Od)+ischemia 44.57 ± 6.40a 0.014 ± O.OO la 1251.61 ± 132.72b
8-03(l 5d)+ischemia 33.66 ± 9.20a 0.015 ± 0.002a 1407.67 ± 142.12b


Values represent mean ± SD. Statistical significance between a and b of at least p<O. 05

The histhological study of the kidney was in accordance with the biochemical findings. Table III shows the percent of kidney without lesions in the different experimental groups. The groups 03-5, 10 and 15 d + ischemia presented higher percent of kidneys without lesions in comparison with the ischemia and 02-5, 10 and 15 d + ischemia groups. Best results were achieved with I 0 and 15 days of ozone treatment before the ischemia.

able III. Results of the histological  study showing the percent of kidneys without lesions.

Experimental  Groups (n=lO) Kidneys without lesions (%)
I -Control






4-02( I Od)+ischemia


5-02( l 5d)+ischemia




7-03( 1Od)+ischemia


8-03(l 5d)+ischemia



No lesions were present in the control group (Figure IA). Kidney samples of the animals subjected to ischemia (Figure IB) showed subcapsular hemorrhage, dilatation of convoluted tubules and of the Bowman ‘s capsule and glomerular compression . The same was achieved in the kidney samples of animals treated with oxygen, during I 0 days, before the ischemia (Figure IC), but in addition, capsules without glomeruli  were found . In contrast, kidney samples of the animals subjected to ischemia with previous ozone treatment (10 sessions) showed minimal lesions which consisted of dilatation of convoluted tubules, preserving the glomeruli and capsule in good shape (figure ID).


Figure 1. Histological study. (A): Control group, no apparent kidney lesions. (B): Ischemia, subcapsular hemorrhage, dilatation of convoluted tubules and of the Bowman ‘s capsule and glomerular compression . (C): 02(10d) + ischemia, the same as ischemia group, but in addition, capsules without glomeruli . (D) 03(10d) + ischemia, minimal lesions which consisted of dilatation of convoluted tubules, preserving the glomeruli and capsule  in good shape. A, B and C (lOOx); D (250x).



It has been demonstrated that a repeated and non-lethal stress is able to confer protection against  a  prolonged  and   severe   stress   (8,10-13,22).  In  these conditions, oxidative preconditioning  is analogous to other phenomena  such as ischemic preconditioning  (23), thermal preconditioning  (24)  and chemical      preconditioning  (25).Ozone  oxidative preconditioning  is  a  somewhat  paradoxical  cellular  mechanism  where  an  induction  of tolerance to 03 and ROS generated by toxic agents is achieved . ROS production, occurring during the ischaemia-reperfusion  phenomenon , seems to be a major mechanism of tissue injury (3-7, 26,27). These results, as previous reports (2,8, 12,13,28,29), support the idea that ozone, being  an  oxidant , could  promote  organ  stress  inducing  an  enhancement  of the endogenous antioxidant defense system, in order to preserve the organ undergoing ischemia . Therefore, the development of tissue injury depends upon the balance between generation of ROS and tissue antioxidant defense mechanism . ROS have been recognize increasingly as

potential mediators of inflammatory cells  injury during glomerulonephritis (30). Our  study have demonstrated that repeated daily administrations of the gas mixture (02 + 03), by rectal insuffiation in rats, generated a sort of tolerance to free radicals released after the induced ischemia reperfusion phenomenon . Nevertheless , taking into account the histological study, best  results were achieved  started from  10 d of 03 treatment . On the contrary, in groups 2(ischemia), 3, 4 and 5 (02-5,10 and 15 d + ischemia, respectively) a significant  cellular damage was documented by functional, biochemical  and morphological  criteria.

The decrease in SOD activity levels, could be associated with their inhibition or inactivation related to ischemia-reperfusion process or free radical attack itself  The significant  stimulation of SOD in the 03 (5,10 and 15 d) + ischemia groups suggests that cellular protection is most likely achieved through the reduction in the availability of superoxide anion. The interaction between hypoxanthine and xanthine oxidase results in generation of superoxide. Once formed, superoxide is rapidly converted to hydrogen peroxide (H202), by SOD and then H202 is converted to water (by glutathione peroxidase) or to water and  oxygen  (by  catalase). However, in the presence of various transition  metals, H202 is rapidly converted to hydroxyl free radical. A delicate balance must, therefore, be maintained among the availability of superoxide, hydrogen peroxide and reduced iron to minimize the highly toxic hydroxyl radical formation. Superoxide can also react with nitric oxide producing peroxynitrite, a highly toxic product. Nitric oxide acts as a potent vasodilator readily degraded by superoxide anion, therefore, antioxidants, particularly SOD (through their ability to scavenge superoxide anion) can maximize the renal protective action of nitric oxide. It have been demonstrated  (31,32) the increase activities of SOD, catalase and glutathione peroxidase after chronic 03 exposure. Moreover, in patients treated with ozone by autohemotherapy, an overexpression of glucose- 6-phosphate dehydrogenase,  glutathione peroxidase  and a reduction  of the lipid peroxidation in plasma were obtained (10). In vitro observations (33) provide evidence that elevated SOD protein level confer resistance against ROS damage in mammalian cells and in vivo experiments with transgenic  mice, expressing enhanced MnSOD protein levels, demonstrated protection against otherwise lethal hyperoxia (34). Administration of SOD or  catalase  was highly effective in attenuating the functional and structural abnormality (proteinuria and glomerular lesions) in rats submitted to a damage induced by puromycin aminonucleoside (35,36). Thus, an increase in the antioxidant enzyme activities produced by the ozone treatment, protect kidney cells against the toxic effect of ROS.

Phospholipase A activation generates lysophospholipids and other metabolites responsible of cellular lysis. The increased phopholipase A activity in groups 2, 3, 4 and 5 (ischemia and oxygen groups) suggests that the enzyme may be partly responsible for the kidney  damage noted in the morphological study. On the contrary, the figures in the ozone groups and control (groups 1, 6, 7 and 8) remained without significance differences, indicating that 03 exerted indirectly a protection against the cellular disruption. The same was obtained in the evaluation of fructosamine, as an indirect measure of the oxidative stress. Groups 2, 3, 4 and  5 with figures significantly higher in comparison with the control and ozone groups, suggesting an accelerate protein glycosylation  (37).


Enhanced antioxidant  enzymes activities induced by ozone treatments provided  kidneys with an effective defense system against the toxic effect to  ROS.  Histological  findings demonstrated that the best results were obtained from 10 days of ozone treatment before the ischemia-reperfusion damage. Therefore, ozone therapy may contribute to minimize the oxidative injuries produced by the oxygen radicals released during an organ transplantation.


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