Increased Production of Nitric Oxide Contributes to Renal Oxidative Stress in Endotoxemic Rat

Overproduction of reactive oxygen and nitrogen species leads to oxidative stress and decreased total antioxidant capacity, which is responsible for high mortality from several diseases such as endotoxic shock. Nitric oxide (NO) produced by inducible NO synthase (iNOS) during endotoxemia is the major cause of vascular hyporeactivity, hypotension and multiple organ failure. In this study, we investigated whether increased production of NO contributes to renal oxidative stress in endotoxemic rat. Saline (4 mL kg , i.p.), endotoxin (Escherichia coli lipopolysaccharide, O111:B4; 10 mg kg , i.p.) and/or selective iNOS inhibitor (1,3-PBIT; 10 mg kg , i.p.) were administered to conscious male Wistar rats and mean arterial blood pressure was recorded at 1, 2, 3 and 4 hr after injection. Nitrite and malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity were measured in the sera and/or kidney homogenates at the end of the experiments. Administration of endotoxin caused hypotension associated with increased systemic and renal nitrite production. 1,3-PBIT prevented these effects of endotoxin at 1 hr after injection of endotoxin. Renal MPO activity was decreased by endotoxin which was not changed by 1,3-PBIT. Endotoxin caused a decrease in MDA levels in the renal tissue, which was prevented by 1,3-PBIT. These data suggest that overproduction of NO by iNOS during endotoxemia decreases renal oxidative stress and that inhibition of iNOS restores total renal antioxidant capacity.


INTRODUCTION
Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. The excessive production of reactive oxygen and nitrogen species (ROS and RNS, respectively) associated with inflammation leads to oxidative stress, which is involved with the high mortality from several diseases such as endotoxic shock [1][2][3][4] . There is a considerable body of evidence for redox imbalance and oxidative stress in endotoxic shock. It has been reported that endotoxemia increases markers of oxidative stress and tissue injury in different animal models and in humans. This effect of endotoxin has been found to be correlated with decreased total antioxidant capacity and blood levels of several antioxidants such as α-tocopherol, retinol, vitamin E, vitamin C and β-carotene in humans with sepsis and septic shock [1][2][3][4] . It has also been reported that although total antioxidant capacity was decreased in patients with sepsis, it was increased in patients with septic shock [5] . Increased xanthine oxidase, superoxide dismutase and glutathione peroxidase activity has also been reported in patients with sepsis suggesting increased production of ROS [6] .
However, malondialdehyde (MDA) (an index for lipid peroxidation) levels were also increased, suggesting that the elevations of these antioxidant enzymes were not so effective as to prevent cellular damage.
Among RNS, nitric oxide (NO) derived from inducible NO synthase (iNOS) is considered to be the major cause for systemic hypotension, vascular hyporeactivity, multiple organ failure and high mortality rate which is associated with septic shock [4][5][6][7] . Although the role of NO is controversial in tissue injury with oxidative DNA damage in endotoxemia, most of the clinical trials demonstrate that iNOS inhibitors may serve as a potentially effective pharmacological agents in alleviating endotoxin-induced decrease in antioxidant capacity and tissue injuries [1,4,7] . Since impairment of renal function during endotoxemia also contributes to multiple organ failure [2] , we investigated whether increased production of NO contributes to renal oxidative stress in endotoxemic rat.
Animals and endotoxic shock model: Male Wistar rats weighing 250 to 350 g were fed with standard chow. They were synchronised by maintenance of controlled environmental conditions throughout the duration of the experiments. The circadian rhythmicity of the animals was entrained by a standardised 12 h light and 12 h dark. All animal experiments were carried out according to the proposal of the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by ethics committee of Mersin University School of Medicine. Conscious rats received either endotoxin (10 mg kg 1 , i.p., sublethal dose) or saline (4 mL kg 1 , i.p.) at time 0 and mean arterial blood pressure (MAP) was measured using the tail-cuff method at 0, 1, 2, 3 and 4 hr. Separate groups of rats were treated with endotoxin alone or in combination with the highly selective iNOS inhibitor 1,3-PBIT (10 mg kg 1 , i.p.) at 1 hr after injection of saline or endotoxin. Rats were sacrificed 4 hr after endotoxin challenge and the blood and kidney were collected. Sera were obtained from blood samples by centrifugation at 18.000 rpm for 15 min at 4° C and stored at -20° C until analyzed for the measurement of nitrite levels. The tissues were homogenized in ice-cold buffer (1 mL) (mM: HEPES 20 [pH 7.5], βglycerophosphate 20, sodium pyrophosphate 20, sodium orthovanadate 0.2, EDTA 2, sodium fluoride 20, benzamidine 10, dithiothreitol 1, leupeptin 20 and aprotinin 10) [8] . Cell debris was removed by centrifugation 18.000 rpm for 15 min at 4° C followed by sonication for 15 sec on ice with 50 µL ice-cold Tris (50 mM, pH 8.0) and KCl (0.5 M). The samples were centrifuged at 18.000 rpm for 15 min at 4° C and then supernatants were removed and stored at -20° C until analyzed for the measurement of myeloperoxidase (MPO) activity and protein, nitrite and MDA levels.

Measurement of renal protein content:
The protein content in the tissue homogenates was determined according to Coomassie blue method using BSA for standard [9] . Briefly, Bradford reagent (200 µL) were added to the mixture of tissue homogenate (5 µL) and distilled water (795 µL). Samples (100 µL) were then pipetted into 96 well microtiter plates and absorbance was measured at 620 nm with a microplate reader (Organo Teknika Microwell System, Holland). Linear regression analysis was used to calculate the protein amount in the tissue homogenates from the standard calibration curves of BSA.

Measurement of serum and renal nitrite levels:
In biological systems conversion of NO in aqueous solution to nitrite and nitrate is thought to favour nitrite production [10] . It has been reported that nitrite is the only stable end-product of the autooxidation of NO in aqueous solution [11] and measurement of nitrite concentrations in the serum and tissue homogenates widely accepted as an index for NOS activity [7,[12][13][14] . Therefore, concentrations of nitrite in serum and tissue homogenates were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production [12] . Briefly, samples (50 µL) were pipetted into 96 well microtiter plates and an equal volume of Griess reagent (1% sulphanylamide (25 µL) and 0.1% N-1-naphtylethylenediamine dihydrochloride (25 µL) in 2.5% ortophosphoric acid) was added to each well. After incubation for 10 min at room temperature, absorbance was measured at 540 nm with a microplate reader (Organo Teknika Microwell System, Holland). Linear regression analysis was used to calculate the nitrite concentrations in the sera and the tissue homogenates from the standard calibration curves of sodium nitrite. Serum and tissue nitrite levels were expressed as µM or mmol mg protein 1 , respectively.

Measurement of renal MPO activity:
MPO is a haem-containing enzyme within the azurophil granules of neutrophils and MPO activity was measured as a simple quantitative method of detecting leukosequestration. The determination of MPO activity as an index of neutrophil infiltration in tissue homogenates depends on the fact that oxidized hidrogen peroxide reduces o-dianisidine [15] . Reduced odianisidine was measured at 410 nm by spectrophotometer. One unit of MPO activity was defined as that degrading 1 µmol of hydrogen peroxide to water per minute at 25° C. Tissue MPO activity was expressed as U mg protein 1 .

Measurement of renal MDA levels:
As an index of lipid peroxidation, the levels of MDA in tissue homogenates were determined by thiobarbituric acid reaction according to Yagi [16] . The method depends on the measurement of the pink color produced by interaction of the barbituric acid with MDA caused by lipid peroxidation. Linear regression analysis was used to calculate the MDA levels in the tissue homogenates from the standard calibration curves of 1,1,3,3tetraethoxypropane. Tissue MDA levels were expressed as mmol mg protein 1 .
Statistical analysis: All data were expressed as means ± SEM. Data were analyzed by one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons and unpaired Student's t or Mann-Whitney U tests when necessary. P value of < 0.05 was considered to be statistically significant.

Endotoxin decreases renal MPO activity:
To investigate the effect of endotoxin on neutrophil infiltration as an index for the development of inflammation, MPO activity was measured in the kidney from endotoxemic animals. Endotoxemia caused a decreased renal MPO activity which was not changed with 1,3-PBIT (Fig. 2). 1,3-PBIT alone had no effect on the basal MPO activity in the tissues (Fig. 2).

Endotoxin-induced increase in systemic and renal nitrite levels is associated with decreased oxidative stress:
To investigate the effect of endotoxin on oxidative stress, one of the products of lipid peroxidation, MDA, was measured in the kidney from endotoxemic animals. Endotoxin decreased renal MDA levels (Fig. 3), which was prevented by 1,3-PBIT. 1,3-PBIT alone had no effect on the basal MDA levels in the tissues (Fig. 3).

DISCUSSION
This study demonstrates that increased systemic NO production by iNOS 4 hr after endotoxin injection causes hypotension in conscious rats and NO-derived from renal tissue also contributes to these effects of endotoxin. More importantly, we showed that endotoxemia-induced increase in NO production suppresses lipid peroxidation in the renal tissue and selective inhibition of iNOS with 1,3-PBIT restores renal antioxidant capacity presumably due to decreased levels of antioxidant molecules or activities of antioxidant enzyme systems.
Expression of iNOS protein and mRNA expression approximately 3 hr after systemic endotoxin administration to animals has been reported in several tissues, such as blood vessels, heart, kidney and lung associated with significant increase in serum/plasma and tissue nitrite levels and decrease in MAP [7,[17][18][19] . In our previous studies with conscious male Sprague-Dawley rats [8,20] , endotoxin (10 mg kg 1 , i.p.) causes a gradual fall in MAP during 4 hr associated with renal iNOS protein expression as well as increased levels of nitrite in serum and kidney which are prevented by 1,3-PBIT (10 mg kg 1 , i.p.; 1 hr after endotoxin). In the present study, administration of endotoxin to Wistar rats also decreased MAP at 4 hr which was associated with an increase in the serum nitrite levels that was prevented after inhibition of iNOS with 1,3-PBIT. Endotoxin-induced increase in the renal nitrite production was also blunted by 1,3-PBIT. These observations suggest that NO produced by activation of iNOS in renal tissue contributes to the endotoxininduced hypotension in conscious rats.  It is well known that oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants [4] . However, there are conflicting reports showing the effect of NO on lipid peroxidation [21,23] and oxidative stress [1,3,4] .
Although increased activity of antioxidant enzymes such as xanthine oxidase, superoxide dismutase and glutathione peroxidase has been reported in patients with sepsis, MDA levels have also shown to be increased, suggesting that the elevations of these antioxidant enzymes are not so effective as to prevent cellular damage [5] . In the present study, endotoxininduced increase in the nitrite levels in serum and kidney was associated with increased renal MDA levels which were prevented by 1,3-PBIT. Therefore, it can be concluded that NO produced by iNOS mediates the endotoxin-induced increase in total antioxidant capacity and consequently decrease in lipid peroxidation. These results are also consistent with the observation that NO behaves as a potent antioxidant [21] .
There are contradictory reports in the literature concerning role of NO as an antiinflammatory or proinflammatory agent [7,17,18] . In the present study, we found that endotoxin-induced increase in the levels of nitrite in serum and kidney which were prevented by 1,3-PBIT was associated with decreased MPO activity. However, decreased renal MPO activity was not reversed by 1,3-PBIT. These results suggest that NO produced by iNOS, expressed in, at least, kidney, does not act as an proinflammatory mediator in this endotoxemia model in rat.
In summary, overproduction of NO during endotoxemia leads to suppression of lipid peroxidation in renal tissue. Selective inhibition of iNOS with 1,3-PBIT restores renal antioxidant capacity presumably due to decreased levels of antioxidant molecules or activities of antioxidant enzyme systems. Impairment of renal function is critically involved in the pathophysiological sequale in septic shock finally resulting in multiorgan failure and death; restoration of these impaired functions should improve therapeutic benefit. Our results suggest that treatment with the selective iNOS could improve the renal function and increased antioxidant capacity in patients during septic shock.