Hyperbaric Oxygen Therapy Alleviates Oxidative Stress and Tissue Injury in Renal Ischemia/Reperfusion Injury in Rats

Abstract

Hyperbaric oxygen (HBO) therapy has been shown to attenuate renal ischemia/reperfusion (I/R) injury in rats, when applied in the early reperfusion period. The aim of this study was to elucidate possible beneficial effects of HBO therapy on renal I/R injury, when applied 24 h after ischemia. Rats were randomized into three groups: (1) control group (n = 20), (2) I/R group (n = 20), and (3) I/R + HBO group (n = 20). Renal I/R injury was created by interrupting renal blood flow for 30 min with a non-traumatic vascular clamp. HBO therapy was administered 24 h after I/R injury and continued for 5 days. At the end of the study, rats were sacrificed under anesthesia, blood was drawn, and right kidneys were harvested for analysis. Renal I/R injury increased serum and tissue malondialdehyde (MDA) levels and reduced superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels. HBO therapy attenuated MDA levels by increasing SOD and GPx activities. HBO therapy also prevented neutrophil infiltration and tissue injury in kidneys. Taken together, HBO therapy has been found to be effective in the delayed period of I/R injury.

• experimental
• hyperbaric oxygen
• oxidative stress
• renal failure

INTRODUCTION

Ischemia is the interruption of blood flow to tissues. Prolonged ischemia causes loss of cellular integrity and eventually cellular death.¹ Renal ischemia has been observed in various clinical situations such as renal transplantation, partial nephrectomy, cardiopulmonary bypass, and various urological procedures. Restoration of blood flow after a period of ischemia may further increase the initial damage via release of oxygen free radicals [reactive oxygen species (ROS)].² Free radicals react with almost all biomolecules including nucleic acids, proteins, lipids, carbohydrates, and connective tissue macromolecules and cause reversible or irreversible changes in the structure of these molecules.³ While small amounts of free radicals are formed during ischemia, much larger amounts are formed in the reperfusion period.⁴ Free radical scavengers are one of the main focuses of current ischemia/reperfusion (I/R) research, but an effective treatment ready to use in routine practice has not been found yet.^2,5,6

Hyperbaric oxygen (HBO) therapy involves the intermittent inhalation of 100% of oxygen under a pressure greater than 1 atm. HBO therapy increases the dissolved oxygen concentration in the arterial blood and enhances the rate of diffusion of oxygen into poorly perfused tissues. Animal studies showed that HBO therapy might protect certain tissues, including muscles, kidneys, liver, heart, and brain against I/R injury.⁷ Rubinstein et al.⁸ and Solmazgul et al.⁹ treated rats with HBO therapy during the early reperfusion period after renal ischemia. They found that HBO therapy protected kidneys against I/R injury.^8,9 In clinical scenarios, HBO therapy may not be readily available, and initial treatment may be delayed. To our knowledge, no one has investigated whether HBO therapy might protect the kidneys against I/R injury even when administered beyond several hours of ischemia.

The aim of this study was to elucidate potential beneficial effects of HBO therapy on renal I/R injury, when administered 24 h after ischemia.

MATERIALS AND METHODS

Animals and Study Groups

This study was approved by Animal Experiments Ethical Committee of Gulhane Military Medical Academy (0530-17-05/482). Sixty male Sprague-Dawley rats, weighting 250–350 g, were used. The animals fed a standard rat chow, drank water ad libitum, and were housed in cages under controlled temperature (20°C–22°C) in 12-h light/dark cycles. The rats were randomly divided into three groups: “control group”, “ischemia group,” and “ischemia + HBO group.” Animals in the ischemia group received left nephrectomy and 30 min of right renal ischemia. Animals in the ischemia + HBO group received HBO therapy for 5 days after left nephrectomy and 30 min of right renal ischemia.

Surgical Procedure

The animals were anesthetized with a combination of ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (10 mg/kg). After midline laparotomy, left nephrectomy was performed. Right renal pedicle was exposed, and blood flow was interrupted for 30 min by applying a non-traumatic vascular clamp to the right renal artery. Clamps were removed and blood flow restored at the end of 30 min of ischemia. Color change was observed in the kidneys for 2 min. Animals were followed for 5 days. At the end of the study, rats were anesthetized [ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (10 mg/kg)], right kidneys were harvested, and blood was drawn for analysis.

HBO Therapy

HBO therapy was administered in an experimental hyperbaric chamber 24 h after the initiation of ischemia. HBO protocol involved 60 min of treatment at 2.5 atm absolute for 5 days.

Measurement of Lipid Peroxidation and Antioxidant Enzyme Activities

Blood and kidney tissue levels of malondialdehyde (MDA) for the determination of the levels of oxidative stress-induced lipid peroxidation, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) for determining the extent of antioxidant response were measured.

The right kidneys of rats were put into tubes, frozen with liquid nitrogen, and stored at −70°C. The frozen kidney tissues were homogenized in a phosphate buffer (pH 7.4) by means of a homogenizer (Heidolph Diax 900; Heidolph Elektro GmbH, Kelhaim, Germany). The supernatant was divided into two to three parts, put in separate tubes, and re-stored at −70°C. The protein content of the kidney homogenates was measured using Lowry’s method, with bovine serum albumin as the standard.¹⁰

MDA was determined by using the method described by Ohkawa et al.¹¹ This method was used to obtain a spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid solution was added to 0.5 mL of homogenate in each centrifuge tube and placed in a boiling water bath for 15 min. The mixture was cooled and centrifuged at 3000 × g for 20 min. Next, 2 mL of the supernatant was added to 1 mL of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled and its absorbance at 532 nm was measured using a spectrophotometer (Helios Epsilon, Unicam, USA). MDA levels were expressed as mmol/g protein.

SOD activity was assessed using the nitroblue tetrazolium (NBT) method described by Sun.¹² The stock solution contained 10 mg of Cu-Zn-SOD from bovine liver dissolved in 10 mL of isotonic saline and was diluted to 600 μg/L with distilled water before it was used in the assay. The SOD assay reagent consisted of a combination of the following reagents: 80 mL of 0.3 mmol/L xanthine solution, 40 mL of 0.6 mmol/L ethylenediaminetetraacetic acid (EDTA) solution, 40 mL of 150 μmol/L NBT solution, 24 mL of 400 mmol/L Na₂CO₃ solution, and 12 mL of bovine serum albumin. The samples were subjected to ethanol and chloroform (62.5% and 37.5%, respectively) extraction prior to the assay of enzyme activity. Briefly, 400 μL of ice-cold ethanol and chloroform mixture was mixed thoroughly with 250 μL of sample. After vortexing for 30 s and centrifugation at 3000 × g at 4°C for 5 min, the upper aqueous layer was collected. The collected supernatant was diluted by a factor of 100, and 0.5 mL of the diluted solution was used for the assay by adding it to 2.5 mL of SOD assay reagent. NBT was reduced to blue formazan by , which has a strong absorbance at 560 nm. One unit (U) of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. The calculated SOD activity was expressed as U/mg-protein.

GPx activity was measured using the method described by Paglia and Valentine, in which GPx activity was coupled with the oxidation of Nicotinamide adenine dinucleotide phosphate (NADPH) by glutathione reductase.¹³ NADPH oxidation was measured spectrophotometrically at 340 nm at 37°C. The reaction mixture consisted of 50 mmol potassium phosphate buffer (pH: 7), 1 mmol EDTA, 1 mmol NaN₃, 0.2 mmol NADPH, 1 mmol glutathione, and 1 U/mL of glutathione reductase. The absorbance at 340 nm was recorded for 5 min. The activity was the slope of the lines as mmol of NADPH oxidized per minute. GPx activity was presented as U/g protein.

Histopathological Examination

A minimum of 20 fields of each kidney section was examined for histopathological examination in 10 magnifications (400×). Histopathological changes were evaluated as tubular cellular swelling (1 point), interstitial edema (1 point), medullary congestion (1 point), medullary ischemia (1 point), and epithelial and hyaline deposition (1 point). The severity of neutrophil infiltration was evaluated between 1% and 100%. A pathologist, blinded to study groups, assessed the samples.

Statistical Analysis

All statistical calculations were performed with a commercial statistical package (SPSS PC, Ver.12.0; SPSS Inc., USA). Numerical values were expressed as mean ± standard error (mean ± SEM). A p < 0.05 was considered to be statistically significant. Mann-Whitney U-test and Student’s t-test were used in comparison of differences between groups.

RESULTS

Oxidative stress parameters in blood and tissue samples are presented on Table 1. MDA, which is the end product of lipid peroxidation, is widely used as a marker of oxidative stress. MDA levels increased in the ischemia group when compared to the control group (p < 0.05). HBO therapy administered after renal I/R injury significantly reduced MDA levels in both serum and tissue samples (p < 0.05). Tissue MDA levels were similar in the control group and ischemia + HBO group. SOD levels decreased in the ischemia group when compared to the control group. HBO therapy restored SOD activity after renal ischemia (p < 0.001). Similarly, GPx levels decreased after renal ischemia and increased both in the serum and tissues after HBO therapy when compared to the ischemia group (p < 0.001) (Table 1).

Table 1. Serum and tissue oxidative stress parameters.

		MDA (mmol/g protein)	SOD (U/g protein)	GPx (U/g protein)
Blood	Control group	0.28 ± 0.02	43.16 ± 0.99	43.78 ± 0.99
	Ischemia group	1.04 ± 0.03^a	16.52 ± 0.34^a	16.66 ± 0.33^a
	Ischemia + HBO group	0.47 ± 0.01^a,b	27.19 ± 1.15^a,b	29.90 ± 1.18^a,b
Tissue	Control group	0.58 ± 0.03	572.95 ± 33.70	35.57 ± 2.48
	Ischemia group	1.51 ± 0.11^a	44.48 ± 4.05^a	15.28 ± 0.59^a
	Ischemia + HBO group	0.66 ± 0.04^b	341.29 ± 1.15 ^a,b	23.46 ± 1.25^a,b

Notes: Data are expressed as mean ± SEM. MDA, malondialdehyde; SOD, superoxide dismutase; GPx, glutathione peroxidase; HBO, hyperbaric oxygen.

^ap < 0.05 versus control group.

^bp < 0.05 versus ischemia group.

Renal ischemia caused epithelial and hyaline accumulation, tubular cellular swelling, interstitial edema, tubular dilatation, medullary congestion, and medullary ischemia. HBO-treated rats showed less tissue damage compared to the ischemia group (Table 2). Considerable neutrophil infiltration was observed in rats exposed to renal I/R injury. Neutrophil infiltration in HBO-treated rats was significantly less compared to the ischemia group (Table 2).

Table 2. Histopathologic score and neutrophil infiltration in kidneys.

	Histopathologic score	Neutrophil infiltration (%)
Control group	0.00 ± 0.00	1.1 ± 0.11
Ischemia group	5.23 ± 0.4^a	23.22 ± 4.35^a
Ischemia + HBO group	2.44 ± 0.3^a,b	14.57 ± 2.54^a,b

Note: Data are expressed as mean ± SEM.

^ap < 0.05 versus control group.

^bp < 0.05 versus ischemia group.

DISCUSSION

HBO therapy has been shown to attenuate I/R injury in certain tissues, including muscles, kidneys, liver, heart, and brain against I/R injury.⁷ Several studies investigated the effects of pre- and post-HBO therapy in experimental I/R injury in rats. Gurer et al.¹⁴ found that HBO prior to ischemia displayed a beneficial effect on renal I/R by reducing oxygen radical peroxidation of lipid membranes. He et al.¹⁵ treated rats with HBO therapy for 2 days before the initiation of renal I/R injury (45 min of ischemia and 24 h of reperfusion). They found that pre-conditioning with HBO protected kidneys against oxidative stress and tissue damage. They also found that HBO increased protein and mRNA levels of heme oxygenase-1 in rat kidneys. In practical terms, however, prediction of the patients who will develop I/R injury is not possible therefore pre-treatment strategies are not useful. Post-treatment effects of HBO therapy have also been assessed in some studies. Rubinstein et al.⁸ treated rats with HBO therapy twice a day after 45 min of renal ischemia. They found that I/R injury reduced glomerular filtration rate and renal blood flow, however HBO attenuated both effects. HBO also reduced lipid peroxidation and increased SOD activity in kidneys. Solmazgul et al.⁹ treated rats with HBO therapy, which was administered in the first 15 min of reperfusion, following 30 min of unilateral renal ischemia. They observed less tubular damage and neutrophil infiltration in HBO-treated rats. Both Rubinstein et al.⁸ and Solmazgul et al.⁹ used HBO therapy in the early period of reperfusion. However, in this study, we administered HBO therapy 24 h after the initiation of reperfusion. We showed that HBO therapy reduced lipid peroxidation and neutrophil infiltration and increased antioxidant enzymes in kidneys. We conclude that HBO therapy is also effective when administered 24 h after I/R injury. In contrast to previous studies we treated rats for 5 days after I/R injury. Hence, one may speculate that the beneficial effects of delayed-HBO therapy observed in our study may be the result of this long-term protocol. Future studies comparing the effectiveness of single and repetitive HBO therapy are needed to address this issue.

Beneficial effects of HBO therapy in renal I/R injury are not fully understood. It has been proposed that HBO affects various factors contributing to the pathogenesis of I/R injury, including neutrophils, endothelium, inflammatory mediators, lipid peroxidation, cellular energetics, and microvascular blood flow.⁷ We have previously shown that HBO therapy reduces neutrophil infiltration after renal I/R injury.⁹ Following ischemia neutrophils increase in human and animal kidneys. Neutrophils activated by the local inflammatory mediators form tight adhesions with endothelial cells, soon after ischemia. Thereafter, these neutrophils infiltrate into tissues and secrete ROS, proteases, myeloperoxidase, and other destructive enzymes that result in tissue damage.^16,17 Therefore, the reduction of the interaction between neutrophils and the endothelium is one of the main objectives of I/R injury treatment.¹⁸ It has previously been shown that HBO reduces these interaction by inhibiting both neutrophil β2 integrin (CD11/CD18) function and intercellular adhesion molecule-1 release on endothelial cells after I/R injury.¹⁹ In this study, we found that HBO therapy reduced neutrophil infiltration after I/R injury and also limited lipid peroxidation by increasing antioxidant enzymes, SOD and GPx. Although the exact mechanisms are not clear, we speculate that beneficial effects of HBO therapy are mediated via inhibiting neutrophil infiltration.

This study has limitations. Serological markers of renal function such as urea and creatinine are lacking.

CONCLUSION

We have shown that delayed-HBO therapy has a protective effect on ischemic acute renal failure in rats. The mechanisms of the beneficial effects of HBO therapy are not fully elucidated. However, the decline in neutrophil infiltration following HBO therapy may have a key role in this pathway. Further studies are needed to better alienate the mechanisms of beneficial effects of HBO therapy in I/R injury.

ACKNOWLEDGMENT

The authors are grateful to Vet. Tayfun Ide, Dr. Turgut Topal and Dr. Tuba Unal for their help in animal experiments, biochemical, and pathological analysis.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

This study was supported by a research grant from Gulhane Military Medical Faculty.