© 1996 American Heart Association, Inc.
Articles |
Role of Endothelium-Related Mechanisms in the Pathophysiology of Renal Ischemia/Reperfusion in Normal Rabbits
the Laboratorio de Nefrologia e Hipertension (C.C., M.R.C., G.P.T., D.T., J.R.M., E.D., M.M., I.M., L.H., S.C., A.L.-F.), Fundacion Jimenez Diaz, Universidad Autonoma, Madrid, Spain; the Universidade Federal de Rio de Janeiro (Brasil) (G.E.); and the Hospital de Mostoles (F.M.), Madrid, Spain.
Correspondence to A. Lopez-Farre, PhD, Laboratorio de Nefrologia e Hipertension, Fundacion Jimenez Diaz, Av. Reyes Catolicos 2, Madrid 28040, Spain.
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Abstract |
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The present study addressed the effect of interventions aimed to increase NO in the setting of acute renal ischemia/reperfusion (I/R) in uninephrectomized rabbits. In the 60-minute post-I/R period, L-arginine+superoxide (O2
-) dismutase (SOD) synergistically improved the renal functional (69.4% versus 10.4% of the pre-I/R glomerular filtration rate with or without L-arginine+SOD, respectively; P<.01) and histological parameters (82.9% decrease of medullary congestion in L-arginine+SOD, P<.01 versus vehicle) and blocked the I/R-dependent neutrophil accumulation (89.3% reduction). In spite of these results over the short term, a second set of experiments disclosed that the protection by L-arginine+SOD was no longer present at 24 and 48 hours (plasma creatinine in vehicle-treated versus L-arginine+SOD–treated animals [mg/100 mL]: 24 hours after I/R, 9.4±1.9 versus 8.07±0.65; 48 hours after I/R, 11.6±3.6 versus 9.7±0.9; P=NS in all the cases). Additional experiments were conducted using a milder 30-minute ischemic model, which showed no significant functional or histological protection by using L-arginine+SOD. In conclusion, our experiments disclosed the following: (1) the critical importance of the interaction between NO and O2- in the acute protective effect of L-arginine (this effect not only improved renal function and histology but also reduced neutrophil accumulation) and (2) the discordance existing between the immediate protection afforded by L-arginine+SOD and the lack of protection observed at 24 and 48 hours. This finding suggests that a punctual intervention on the NO system at the time of I/R is not sufficient to reduce renal damage over the long term.
Key Words: L-arginine • superoxide dismutase • neutrophil • nitric oxide
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Introduction |
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Ischemia/reperfusion is a leading cause of organ damage in diverse clinical circumstances (for review, see References 1 through 3). The mechanisms proposed to explain the I/R-induced injury include anoxia, release of oxygen-derived free radicals during reperfusion, neutrophil accumulation, and the subsequent release of additional oxygen-derived free radicals and lytic enzymes.1 2 3 Several approaches have been tried against the above-mentioned damaging mechanisms, aimed to reinstigate blood flow by using vasodilatory drugs,4 reduce oxygen-derived free radical effects by means of scavenging agents,5 6 and diminish neutrophil accumulation or block the activation of neutrophil-borne tissue-damaging enzymes, by using antibodies against adhesive integrins or protease inhibitors.7 8
In recent years, considerable information has been gathered on the role of the L-arginine metabolite NO in circulatory regulation (for review, see Reference 9). Even though hypoxia is supposed to create profound alterations in endothelial cell function, data about the role of NO and NO-related mechanisms in the setting of renal I/R are still scarce.10 11 12 13 14 Basically, in the late 1980s, a decreased endothelium-dependent vascular relaxation was demonstrated in the postischemic period.10 11 More recently, a decreased NO production has been related to an impaired kidney functional outcome after I/R.13 14 However, although these data suggest a potentially beneficial role of NO in counteracting the immediate complications of I/R, more information is still necessary to provide definitive support to this hypothesis. Moreover, major technical difficulties and potential changes in the predicted outcome of I/R are posed by the interaction between NO and O2- to generate the free radical peroxynitrite,15 16 17 18 19 which has a marked damaging potential through the production of OH-.
Therefore, the aim of the present study was to analyze the role of NO-dependent mechanisms in the renal response to I/R. The working hypothesis was that providing a source of NO might have beneficial effects on both I/R-related renal functional derangements and neutrophil accumulation, provided NO is protected from interacting with O2-. For this purpose, we analyzed the effect of the NO precursor L-arginine and the NO donor sodium nitroprusside in animals submitted to renal I/R, both in the presence or in the absence of SOD. The study analyzed the effects of the above-mentioned maneuvers on renal functional parameters, morphology, and neutrophil accumulation. Further experiments have been performed comparing the effects of L-arginine+SOD at early (1-hour) and late (24- and 48-hour) post-I/R periods to assess the overall effectiveness of this combination on the post-I/R renal outcome. In additional studies using 30-minute clamping of the renal artery, we analyzed whether the effects of L-arginine+SOD were related to the severity of the ischemic insult.
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Materials and Methods |
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Animal Instrumentation and Experimental Procedures
The study protocols were approved by the institutional review board and performed according to the international conventions on animal experimentation. All the studies were performed in male New Zealand rabbits, weighing 2.5 to 2.8 kg. All the animals had free access to food and water. After sodium pentobarbital anesthesia (100 mg/kg body wt IP), rabbits underwent femoral vein and artery cannulation, and the abdominal cavity was opened with a midline incision. A continuous PSS (mmol/L: NaCl 137, KCl 2.6, KH2PO4 1.5, NaH2PO4 8, and glucose 5.6, pH 7.4, 37°C, 0.25 mL/min) infusion was started and maintained throughout all the experiment. Next, right uninephrectomy was performed, the first branch of the renal artery and ureter were cannulated, and heparin (100 IU/kg body wt IV) was administered. The extracted kidneys were frozen in liquid nitrogen to serve as baseline controls of MPO activity. Mean arterial pressure was continuously monitored (Letica polygraph, Scientific Instruments), and body temperature was monitored by an intrarectal electrical thermometer (Elektrolaboratoriet) and maintained between 36°C and 37°C to avoid possible interferences in the ischemic phenomenon by temperature changes.20 At the end of a 45-minute equilibration period, blood samples were taken for determination of packed cell volume, creatinine, and electrolytes. Thereafter, the left renal artery was occluded for 60 minutes with a nontraumatic clamp, as described previously.21 At the end of this time period, the clamp was released, and reperfusion was allowed for an additional 60 minutes. Once the reperfusion phase was finished, the animals were killed by an overdose of anesthesia, and the kidney and heart were extracted, washed with cold PSS, and frozen in liquid nitrogen.
To further examine the prolonged effects of the pharmacological maneuvers on I/R, additional groups of rabbits were studied at 24 and 48 hours after I/R. In these animals, the experimental procedures were similar to those described above, but instead of killing them at 60 minutes after I/R, the rabbits were maintained alive in metabolic cages with free access to food and water and with a constant 22°C temperature from 48 hours before to 48 hours after I/R. Urine was collected during surgery and I/R through a transitory bladder catheter.
Finally, to determine whether the effects of the L-arginine+SOD infusion were conditioned by the severity of the ischemic insult, two additional groups of rabbits were studied after clamping the renal artery for only 30 minutes. All the experimental procedures used in these animals, with the exception of the time required for clamping, were identical to those of the groups mentioned above.
The two latter sets of experiments were performed in view of the results obtained in the animals studied at 1 hour after 60 minutes of I/R. Accordingly, a simplified protocol was used to reduce animal manipulation to the minimum necessary to fulfill the specific aim of this particular experiment, ie, to assess the protective efficacy of L-arginine+SOD in a longer term follow-up. In the 60-minute I/R animals, samples were obtained for the determination of blood and urinary electrolytes and creatinine at baseline and at 24 and 48 hours after I/R. In the last group studied (30-minute I/R), blood was obtained only at 48 hours after completing the surgical procedure to minimize the putative stressing factors acting on the animals. In all the cases, the animals were killed by an overdose of anesthesia.
Administration of the Agents
All the infusions were freshly prepared in sterile PSS (37°C, pH 7.4) immediately before using. The different agonists were given, as detailed below, by continuous intrarenal infusion (3.75 mL/h) through the retrograde cannulated branch of the renal artery. All the infusions followed a similar schedule, beginning immediately before (5 minutes) the initiation of reperfusion and lasting during the entire reperfusion phase (total volume infused, 4.1 mL). In the animals receiving no pharmacological agents, a similar volume of the vehicle was administered.
Measurement of Renal Function
Renal functional parameters were measured as previously described,21 22 by using methoxy-[14C]IN (loading dose, 3 µCi/mL; infusion, 0.62 µCi/mL in PSS, 3 mL/h) and [3H]PAH (loading dose, 13 µCi/mL; infusion, 2.5 µCi/mL, 3 mL/h). Two 30-minute urine collections were completed, with blood sampling (150 µL) at the beginning and end of each clearance period. 3H and 14C activities were measured using a two-channel liquid scintillation counter, which corrects for the interference between isotopes. CIN was used for calculating FENa. Even though important changes in the renal handling of PAH may occur during ischemia, CPAH was used as a comparative index of the changes occurring with each experimental maneuver. The absolute values of CPAH should, however, be considered with the caveat that I/R may alter the actual magnitude of this variable. Even with these theoretical concerns, these data are worthy of consideration as an additional comparative marker of renal functional improvement and probably of increased renal blood flow. Electrolytes were measured by an automatic analyzer (Astra IV, Beckman).
Experimental Protocols
To assess the role of the L-arginine/NO–mediated vasodilator system in the renal response to a 60-minute I/R, we studied the effect of the following intrarenal infusions: (1) L-arginine alone (0.5 mg/kg body wt per minute, n=8); (2) SOD alone (2.2 U/kg body wt per minute, n=8); (3) combined L-arginine (0.5 mg/kg body wt per minute) and SOD (2.2 U/kg body wt per minute) (n=12) (the latter combination was used to avoid the interaction of NO and O2-, with the subsequent production of peroxynitrite; see below); (4) sodium nitroprusside (3 µg/kg body wt per minute, n=6), used as an NO donor, which has been shown in in vivo or entire-vessel experiments to be less affected or not affected by O2-23 24 25 (to directly ascertain whether or not the effect of sodium nitroprusside was influenced by O2- in the present experimental conditions, a complementary study [n=4] associating sodium nitroprusside at 3 µg/kg body wt per minute and SOD at 2.2 U/kg body wt per minute was performed; in these and all the experiments using sodium nitroprusside, the preparation was permanently protected from the light); (5) dopamine (2 µg/kg body wt per minute, n=4), a non–NO-mediated vasodilator, used to assess whether any effect of the maneuvers mentioned above (items 2 to 4) could be unspecifically attributed to vasodilation rather than to NO production (in previous setting-up experiments, we found that in control rabbits this dose of dopamine increased CIN and CPAH by 30±2% [P<.01] and 47±3% [P<.005], respectively [M.R. Cernadas et al, unpublished data, 1996]).
As mentioned before, additional groups were studied as follows: (1) uninephrectomized rabbits submitted to 60-minute I/R and infused with vehicle alone, (2) identically instrumented rabbits, treated with an infusion of L-arginine+SOD, similar to the infusion 3 group above, (3) uninephrectomized rabbits submitted to 30-minute I/R and infused with vehicle alone, and (4) identically instrumented rabbits, treated with an infusion of L-arginine+SOD, similar to the infusion 3 group above.
Pathology Studies
Kidney samples from control, ischemic, and ischemic L-arginine+SOD–treated animals were fixed in 10% formaldehyde, stained with routine techniques, and examined by optical microscopy in a blinded fashion by a renal pathologist (F.M.). The kidneys were specifically analyzed for the presence of tubular damage and medullary congestion patterns, as suggested by Solez et al,26 and classified using a numerical score as follows: for the degree of tubular damage, 0 indicates no damage; 1, unicellular patchy isolated necrosis; 2, tubular necrosis <25%; 3, tubular necrosis between 25% and 50%; and 4, >50% tubular necrosis and the presence of infarcted tissue; for the degree of medullary congestion, 0 indicates no congestion; 1, vascular congestion with identification of erythrocytes with x400 magnification; 2, ibidem, with x200 magnification; 3, ibidem, with x100 magnification; and 4, ibidem, with x40 magnification.
Measurement of Urinary Nitrites (NO2s)
Concentrations of urinary NO2s were determined by the Griess method. Briefly, aliquots of urine were mixed with an equal volume of Griess reagent (1% sulfonamide/0.1% N-1-naphthylenediamine dihydrochloride/3% phosphoric acid) and incubated at 60°C for 30 minutes. The absorbance at 554 nm was measured by a flow-through visible spectrophotometer (model U-2000, Spectrophotometer Ltd).
Measurement of MPO Activity
MPO activity was measured in animals submitted to I/R with or without the different maneuvers detailed in the experimental protocols by assessing the change in absorbance of o-dianisidine hydrochloride, as described previously,27 28 and after homogenizing the tissues in 50 mmol/L potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide. In the conditions used, no interference might be expected from peroxidase activities other than MPO.28 A standard in vitro curve was constructed using known numbers of neutrophils, which showed a significant correlation between neutrophil number and MPO activity (r=.92, P<.001).
Statistics
Values are expressed as mean±SEM. Unless stated otherwise, all the results correspond to a minimum of five experiments. Changes in variables within the same group and between groups were analyzed by one- and two-way ANOVA and subsequent Scheffe's and Fisher's tests. Differences between two groups were evaluated by the unpaired Student's t test, and differences between two periods of the same animals were evaluated by the paired Student's t test. A value of P<.05 was considered significant.
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Results |
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Studies at 1 Hour After 60-Minute I/R
Renal Hemodynamics and Functional Studies
The occlusion of the renal artery for 60 minutes in control uninephrectomized rabbits induced a decrease of CIN, CPAH, and UV and an increase of FENa in the first hour after unclamping of the renal artery (P<.001 with respect to the baseline) (Table 1
and Fig 1). For all these studies, the baseline values of the I/R group were used for comparison, since the baseline measurements were statistically identical in all the groups (data not shown). In the animals receiving the combined L-arginine+SOD infusion, both CIN and CPAH were significantly restored (Table 1). On the other hand, the separate administration of L-arginine or SOD did not affect the post-I/R decrease in CIN and CPAH (Table 1, P=NS with respect to I/R alone). A significant recovery of the post-I/R decrease in CIN and CPAH, in the range of that observed with L-arginine+SOD infusion, was obtained with sodium nitroprusside (Table 1). None of the experimental maneuvers provoked significant changes in systemic mean arterial pressure (98±5.4, 96.5±4.1, and 95.8±4.6 mm Hg in control, L-arginine+SOD, and sodium nitroprusside groups, respectively). The UV was significantly decreased (Fig 1, top left) in the post-I/R period, although a simultaneous increase in FENa was observed (Fig 1, bottom). The infusion of L-arginine+SOD or sodium nitroprusside markedly increased both diuresis and natriuresis (Fig 1, top left and right) and decreased FENa (Fig 1, bottom). None of the above-mentioned changes was observed with L-arginine or SOD alone (Fig 1). The values of the above-mentioned variables obtained with the addition of SOD to sodium nitroprusside infusions did not differ from those with sodium nitroprusside alone (P=NS; data not shown). The experiments using dopamine demonstrated no effect on CIN and a moderate effect on CPAH (Table 1). A significant, albeit moderate, effect on UV (50.9±5.4 µL/min, P<.05 with respect to I/R alone) and UNaV (9.9±1.8 µEq/min, P<.02 with respect to I/R alone) was also detected in the dopamine-treated animals. The FENa was, however, in a range similar to that found after I/R (8.9±0.9%, P=NS).
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Renal Histology
As shown in Figs 2 and 3, the combination of L-arginine+SOD significantly reduced vascular medullary congestion and tubular damage. Scores at baseline are not represented, since they were not different from zero. No significant protective effects occurred with L-arginine or SOD alone (P=NS with respect to I/R alone; data not shown). No significant neutrophil accumulation was found at optical microscopy with any of the experimental maneuvers (P=NS for all the samples with respect to the nonischemic controls; data not shown).
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Measurement of Urinary Nitrites
Values of urinary nitrites are shown in Table 2. As can be observed, I/R drastically affected nitrite excretion, which decreased to levels below the threshold of the method. The use of L-arginine+SOD infusions significantly restored urinary nitrite excretion. Neither L-arginine nor SOD alone produced a relevant increase of nitrite excretion with respect to the values of I/R+vehicle (16.8±4.5 and 7.3±2.5 nmol/h, respectively; P=NS with respect to the values with I/R; n=3). Of further interest, the administration of dopamine did not increase the nitrite excretion with respect to I/R, in spite of the dopamine effects on UV (3.5±1.2 nmol/h, P=NS with respect to I/R).
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Measurement of MPO Activity: Ischemia/Reperfusion-Induced Neutrophil Accumulation and Role of the L-Arginine/NO Vasodilator System
An increased renal MPO activity, a specific marker of neutrophil accumulation, was observed after I/R (Fig 4, left). This increase was abolished by the combination of L-arginine+SOD or by sodium nitroprusside but remained unchanged with L-arginine alone; SOD alone produced only a minor reduction of post-I/R MPO activity (Fig 4). The renal MPO activity detected with the coinfusion of sodium nitroprusside+SOD did not differ from the values with sodium nitroprusside alone (0.31±0.02 U/g tissue, P=NS with respect to sodium nitroprusside alone). No significant changes in baseline MPO activity were observed by the administration of L-arginine, SOD, or sodium nitroprusside in sham-operated animals that were submitted to a similar protocol, but without the induction of I/R (P=NS, n=3 each; data not shown). An increased MPO activity was also detected in the hearts of the animals with renal I/R (Fig 4, right). This phenomenon was inhibited in the presence of L-arginine+SOD or sodium nitroprusside but not with L-arginine; however, SOD alone produced a partial, albeit significant, decrease (Fig 4, right). No changes in MPO activity were observed in either the kidney or the heart in sham-operated rabbits (MPO activity, 0.47±0.01 U/g kidney tissue and 0.17±0.04 U/g heart tissue; n=3; P=NS).
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Studies at 24 and 48 Hours After 60-Minute I/R
Rabbits With 60-Minute I/R
At 48 hours after I/R, the kidneys had a massive histological damage (degree 4, ie, generalized tubular necrosis with significant areas of infarction). Contrary to the findings in the aforementioned 1-hour experiments, no differences were found in this histological pattern between vehicle-treated (n=3) and L-arginine+SOD–treated (n=4) animals. In the same regard, no differences between both groups were detected in functional variables at 24 and 48 hours. With this severe model of renal injury, the animals produced urine only during the first 60 to 90 minutes, in amounts similar to those described in the experiments in the above section (respective values for vehicle-treated versus L-arginine+SOD–treated animals: UV, 7.5±0.8 and 101.3±22.8 µL/min; UNaV, 5.7±0.8 and 10.6±2.3 µEq/min; P<.01 between both groups). However, during the following 48-hour follow-up period, the rabbits remained practically anuric. Accordingly, no significant differences were detected in plasma creatinine (respective for vehicle-treated versus L-arginine+SOD–treated animals [mg/100 mL]: baseline, 1.25±0.05 versus 1.32±0.16; 24 hours after I/R, 9.4±1.9 versus 8.07±0.65; and 48 hours after I/R, 11.6±3.6 versus 9.7±0.9; P=NS in all the cases) or UV (respective values for vehicle-treated versus L-arginine+SOD–treated animals: 52.5±3.5 and 62.7±5.9 mL/24 h at baseline; 2.8±2.2 versus 2.3±1.9 mL during the 48-hour follow-up; P=NS between both groups). Also, no significant differences appeared in plasma electrolytes (data not shown), with the exception of plasma K+, which was more increased in the L-arginine+SOD group (respective values for plasma K+ in vehicle-treated versus L-arginine+SOD–treated animals: at 24 hours, 4.7±0.3 versus 6.0±0.5 mmol/L; at 48 hours, 5.3±1.6 versus 7.0±1.9 mmol/L; P<.05 and P=NS, respectively).
Rabbits With 30-Minute I/R
The results at a 48-hour follow-up of the animals submitted to 30 minutes of clamping of the renal artery are shown in Table 3. Values at baseline were similar to those of the other groups (data not shown). As expected, the renal functional and anatomic damage in these animals was milder than in the rabbits that were submitted to 60 minutes of I/R. However, the main result was that (as can be observed in Table 3) no improvement was detected because of the treatment of the animals with L-arginine+SOD. In fact, a significant increase in plasma creatinine and a decrease in creatinine clearance were observed in the L-arginine+SOD–treated group (Table 3). Also, a higher value of plasma K+ was found in the L-arginine+SOD–treated group (respective values for plasma K+ in vehicle-treated versus L-arginine+SOD–treated animals at 48 hours: 5.0±0.4 versus 6.5±0.8 mmol/L, P<.05).
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Discussion |
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The present series of experiments addressed the role of interventions aimed to increase NO on the renal response to I/R injury. The rationale of our approach was based on previous evidence showing a decreased response to NO-mediated vasodilators in renal ischemia.1 2 15 16 The results obtained in the short-term observations supported the hypothesis that interventions on NO-dependent mechanisms could modify both the functional and anatomic outcome of ischemic renal failure and the intensity of neutrophil infiltration. However, these effects of L-arginine appeared to be confined to the immediate post-I/R period; ie, this protective effect was no longer evident at 24 and 48 hours after I/R.
Effects of L-Arginine±SOD on the Immediate (60-Minute) Renal I/R Injury
In spite of their putative beneficial effect, the previous experience with the use of amino acids in I/R has been unsatisfactory; actually, according to some reports, amino acids may increase rather than decrease renal damage.2 29
In recent years, the concept has emerged that not all the amino acids are equal regarding the mechanism of their renal effects.30 31 In normal rats, we have observed that the renal action of L-arginine is almost completely related to NO generation.30 The effect of L-arginine is of outstanding interest in the setting of renal I/R. However, recent communications are contradictory about the positive or negative role of L-arginine in acute myocardial ischemia.32 33 In the same regard, data involving the use of L-arginine in renal ischemia were not favorable and showed either no effects13 or minor positive effects.14
The reaction between NO and O2- leading to peroxynitrite generation15 16 17 18 is critical for the properties of NO or NO-donor agents in the setting of increased oxygen-derived free radical production, as occurs in I/R. The reaction between NO and O2- was prevented in our experimental design by the administration of SOD together with L-arginine. Even though a partial protection against I/R-induced damage has been reported with the use of SOD or other free radical scavengers, the literature is far from unanimous on this particular subject.3 4 5 6 34 35 36 A main finding of the present study was that the combination of L-arginine+SOD had a synergistic effect in reversing the post-I/R functional and structural derangement. The fact that SOD had such a marked effect in modifying L-arginine action strongly supports the importance of extracellularly released free radicals in the renal outcome after I/R. This is further illustrated by the finding that L-arginine+SOD but not L-arginine alone increased urinary nitrites, suggesting that the supplementation with an exogenous substrate, namely, L-arginine, needs additional factors to be effective, eg, GFR and renal flow preservation.
The similarity of the results using the combination of L-arginine+SOD or sodium nitroprusside further suggested that the observed effects were due to the action of NO; furthermore, these results showed, for the first time, the critical in vivo importance of eliminating O2- when an increase of NO via L-arginine is attempted. The fact that sodium nitroprusside was effective in I/R conditions and that there was an absence of changes by SOD on sodium nitroprusside effects is most probably due to the fact that sodium nitroprusside releases NO in an intracellular domain with lesser exposure to the reaction with extracellular O2-.37 The differences between our results and those of Shaw et al,38 who found no protection against renal ischemia by using this drug, are related to major methodological differences; ie, in our experiments, sodium nitroprusside was administered intrarenally and before clamp release.
In the same regard, other studies have shown that the improvement of postischemic GFR by oxygen-derived free radical scavengers was not accompanied by a simultaneous amelioration of the histological pattern and Na+ reabsorption on the day after ischemia.39
Even though the prevalent opinion indicates that neutrophil accumulation has a significant role in aggravating the severity of myocardial and mesenteric ischemia,40 41 42 43 the pathogenetic role of neutrophil accumulation in renal I/R is still controversial. Data that either deny7 44 or favor8 45 the importance of neutrophils in renal I/R have been obtained, and the debate is still alive. In our experiments, a significant neutrophil accumulation occurred in the kidney immediately after I/R. This phenomenon was precociously evidenced by the MPO method; at this time, the histological techniques appeared to be less sensitive. The same maneuvers that interfered with the renal functional consequences of I/R (namely, L-arginine+SOD or sodium nitroprusside) blocked neutrophil accumulation. The association of L-arginine+SOD as well as sodium nitroprusside predictably acted by increasing NO levels46 ; NO has been shown to be a relevant anti-neutrophil adhesion molecule in ischemic situations.47 Furthermore, L-arginine can block several manifestations of neutrophil activation.27 Of additional interest are the data showing an increased neutrophil accumulation in the hearts of rabbits with renal I/R. These results suggest that myocardial effects might potentially occur during ischemic episodes in distant organs. Preliminary data from Atanasova et al12 have suggested that in circumstances of NO-generation inhibition, renal I/R may provoke systemic circulatory complications. Moreover, the fact that the increase of myocardial MPO was blocked by the intrarenal L-arginine+SOD infusion suggests that the combination of L-arginine+SOD acts in a significant proportion by inhibiting the I/R-related activation of neutrophils within the reperfused kidney.
Effects of L-Arginine+SOD on the Outcome of Renal I/R Injury at 24 and 48 Hours
A marked difference was observed between the effects of L-arginine+SOD at 1 hour compared with longer periods of observation, which advises against straightforwardly considering L-arginine+SOD as an effective anti-I/R therapeutic agent. This lack of long-term protection was evident not only for the 60-minute ischemic model but also for the lower intensity 30-minute ischemic model. The finding of the causes of this discrepancy between short-term and long-term observations is beyond the scope of the present research, although the design of a valid anti-I/R alternative is of critical importance. However, in a first analysis, the hypothesis can be raised that the administration of L-arginine+SOD was a time- and dose-limited event, which was not capable of overcoming the continuous production of tissue-injuring radicals beyond the time of a short half-life action. This limitation could be theoretically avoided by using a prolonged administration schedule of the NO precursor or NO donors. An alternative, albeit discouraging possibility, is that the maintenance of renal blood flow and GFR in the first hours after the ischemic insult offers no advantage, because tubular damage cannot be sustainedly prevented. Similar results showing a difference between the positive short-term and null long-term effects of SOD in improving myocardial ischemia have been found by other groups.48 49 On the other hand, Lopez-Neblina et al50 have recently communicated that in a rat model of renal I/R, the increase in serum creatinine, the histological damage, and the neutrophil infiltration could be prevented in a prolonged fashion by the administration of sodium nitroprusside before reperfusion.50 These observations are somewhat difficult to interpret, in view of the very short half-life of sodium nitroprusside. In fact, since the most powerful protective effect happened by administrating sodium nitroprusside 75 minutes before reperfusion and since no effects were obtained when it was given 5 minutes before reperfusion, it would be necessary to assume that a modification in the leukocyte response occurred that needed a long time to develop. As stated by the authors,50 this effect requires further characterization to be more completely understood.
The present results induce us to be extremely cautious when evaluating the success of the early protective effects of I/R. On the other hand, since the effects observed after 60 minutes were frankly encouraging in terms of renal protection, an effort should be made to obtain more persistently beneficial results by interventions that will increase NO.
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Acknowledgments |
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This study was supported by grants 0232/93 of Fondo de Investigaciones Sanitarias and PM 92/0041 of the Direccion General de Investigaciones Cientificas y Tecnica, Fundacion Renal Inigo Alvarez de Toledo (FRIAT), and Fundacion Ramon Areces. Dr Mosquera was the recipient of a fellowship from FRIAT, and Drs Cernadas and Monton were recipients of fellowships from Fundacion Conchita Rabago. The authors wish to thank Paloma Tramon and Pilar Manzano for technical assistance.
Received March 22, 1996; accepted August 12, 1996.
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References |
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