nitric oxide production during the hyperacute vascular rejection

JOURNAL OF SURGICAL RESEARCH 63, 375–380 (1996)ARTICLE NO. 0279

Nitric Oxide Production during the Hyperacute Vascular Rejection

JULIO CASTILLO PH.D.,* JOSE R. BERRAZUETA PH.D.,† LUIS HERRERA PH.D.,* JOSE F. VAL-BERNAL PH.D.,‡JAVIER GOMEZ M.D.,‡ MARIA J. SANCHEZ PH.D.,§ ALBERTO RIESTRA M.D.,† MARIA T. GARCIA-UNZUETA M.D.,†

JOSE A. AMADO PH.D.,† EDUARDO SALAS PH.D.,† AND MANUEL GOMEZ-FLEITAS PH.D.*

Departments of *General Surgery, †Internal Medicine, ‡Pathology, and §Cell Biology, Hospital Universitario‘‘Marques de Valdecilla,’’ Avda de Valdecilla s/n, Santander 39005, Spain

Submitted for publication September 6, 1995

Our experimental research tries to determine if oneA complete understanding of the biological mecha- recently described biological mediator, the nitric oxide

nisms that take part in the hyperacute vascular rejec- (NO), could play a role as an effector mechanism duringtion is necessary to prevent its development. Nitric the hyperacute vascular rejection.Oxide (NO) release by the graft could be one of those NO is an endogenous immunomodulator, generatedmechanisms as cytokine induction of NO synthase in in a constitutive way (constitutive/Ca2/-dependent NOreticuloendothelial cells produces NO with cytotoxic synthase) from L-arginine by vascular endothelial cellsactivity. We have performed 20 heterotopic pig-to-dog that is responsible for the stimulation of the solublekidney xenotransplantations in order to determine if guanylate cyclase in different tissues and that it wascytotoxic NO is released during the hyperacute rejec-

identified as the previously known endothelium de-tion. Nitrites and cGMP levels were determined inrived relaxing factor (EDRF) [7, 8]. Furthermore, NO isplasma samples taken from the graft artery and veina cytotoxic molecule released by activated macrophagesuntil vessel thromboses. NO synthase activity wasfollowing immunological challenge. This toxic NO ismeasured in pieces from renal artery and renal tissue.not constitutive, and its generation depends on the in-The results show that after discordant xenotransplan-duction (by immunological stimuli) in the reticuloendo-tation, inducible NO synthase is activated in the trans-thelial cells of a different class of NO synthase (induc-planted organ, indicating that NO can mediate hostible/Ca2/-independent) [9–11]. Even though this sug-tissue damage during the hyperacute rejection. q 1996

gests the possibility that NO can mediate host tissueAcademic Press, Inc.

damage, there is no experimental work in the medicalliterature dealing with this issue.

INTRODUCTION

METHODSDuring the past years, the growing shortage of hu-

man organs for clinical transplantation has led to an An in vivo animal model of discordant xenotransplantation wasused. Twenty heterotopic pig-to-dog kidney xenotransplantationsimportant increase in the xenotransplantation re-were performed.search. Even though some experiments using grafts

Minutes after the organ harvesting, the kidneys from adult ‘‘Large-from closely related species have obtained promisingWhite’’ pigs (40–60 kg) perfused with Collins solution were implanted

results [1–3], the future of xenotransplantation seems in the right iliac vessels of two adult dogs (30–40 kg). Blood samplesto be placed in the use of organs from widely divergent (10cc) were taken from the graft renal vein (minutes 0, 5, 10, and 20

after reperfusion) and artery (minutes 0, 30, 60, 90, and 120) until thespecies, mainly the pig [4]. However, discordant xeno-vessel thromboses. The donor kidney was removed after the renal veintransplantation gives way to a vigorous hyperacute re-thromboses (Group A; n Å 10) or after the renal artery thrombosesjection which results in the graft destruction within(Group B; nÅ 10). Six kidney pig autotransplantation experiences were

minutes of its reperfusion [5]. To improve the xenotrans- used as a control group (Group C; n Å 6). These grafts were removedplantation results, it is imperative to understand the after 30 min (C1; n Å 3) or 120 min (C2; n Å 3).

Blood samples were collected in tubes with heparin or K3 EDTAbiology of the hyperacute vascular rejection.and then centrifuged and stored at 0407C for later determinations.Binding of preformed natural host antibodies to theirPlasmatic Nitrites, the initial products of nitric oxide metabolism,epitope on the donor endothelial cells leads to the acti- were measured using an automated colorimetric method based on the

vation of different cell systems and biological mecha- Griess reaction [12]. Cyclic GMP levels were determined by specificnisms that are responsible for the hyperacute rejection radioimmunoassay [13].

Pieces from the renal artery and the renal parenchyma were frozendevelopment [6]. Some of the former mechanisms, suchseparately in liquid nitrogen for later determination of NO synthaseas the complement and coagulation systems, are wellactivity. This was measured by the formation of radiolabeled 14Cknown, but the understanding of others (endothelial Citrulline from 14C L-arginine in the presence or absence of either

cell activation) is only beginning and there could be EGTA or EGTA plus L-NMMA to determine the level of the constitu-tive and inducible NO synthase activities, respectively [14].more, unknown at the present time.

375 0022-4804/96 $18.00Copyright q 1996 by Academic Press, Inc.

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376 JOURNAL OF SURGICAL RESEARCH: VOL. 63, NO. 2, JULY 1, 1996

Results are expressed as mean { SEM. The paired Student’s t test xenotransplantation was completed. The Inducible ni-and the ANOVA test are used to express the results and to determine tric oxide synthase activity (0.22 { 0.09 pmol/mg/min)the statistical significance.

was greater than the constitutive NO-s activity (0.18 {0.13 pmol/mg/min). There were significant differences

RESULTS between the NO synthases levels at 30 and 120 min,when these activities had decreased to 0.1{ 0.04 pmol/Hyperacute rejection was macroscopically patent amg/min (P Å 0.009) and to 0.053 { 0.04 pmol/mg/minfew minutes after the graft reperfusion. Renal vein(P Å 0.006), respectively. Constitutive NO synthase ac-blood flow stopped after 30 min (33 { 16 min). Renaltivity in the renal parenchyma-autotransplantationartery thromboses was completed after 120 min (110group at 30 min (0.15 { 0.05 pmol/mg/min) and at 120{ 34 min). Hyperacute rejection was confirmed duringmin (0.05 { 0.02 pmol/mg/min) was not different fromthe pathological examination of the organs.the xenotransplantation group (Fig. 4). However, thereNO20 basal levels in the renal vein (1.31{ 0.69 pmol/was no inducible NO synthase activity in the auto-ml) increased during the hyperacute rejection period,transplantation experiments.reaching the maximum level (4.05 { 1.09 pmol/ml) 5

Inducible NO synthase activity (Fig. 5) was almostmin after the surgical procedure was completed andundetectable in the renal artery at 30 min (0.004 {the organ reperfused (Fig. 1). This increase was sig-0.11 pmol/mg/min) and it was completely absent at 120nificant (P Å 0.00001). After reaching this peak, themin. There were no significant differences between theNO20 levels progressively decreased to a minimum ofconstitutive NO synthase activities in the renal paren-1.92 { 1.34 pmol/ml, just before the vein thromboses.chyma (0.18 { 0.13 pmol/mg/min) and the renal arteryThere were no significant differences between the ini-(0.089 { 0.22 pmol/mg/min) at 30 min.tial and the final values (P Å 0.09). Considering the

There was no significant increase or decrease of theglobal analysis of the data presented (ANOVA test),basal cGMP renal vein levels (9.09 { 4.08 nmol/ml)there were significant differences in the NO20 levels induring the xenotransplantation (P Å 0.1145) (Fig. 6).the renal vein during the hyperacute vascular rejection

(P õ 0.0000001). There was no significant increase orDISCUSSIONdecrease of the basal NO20 renal vein levels in the con-

trol groups.NO20 basal levels in the renal artery (1.57 { 0.72 Organ shortage is limiting the present and the future

perspectives for the clinical transplantation. Waitingpmol/ml) remained unchanged during the first 60 minof the hyperacute vascular rejection period, as there lists for this procedure are growing each day, the same

that the mortality rate associated with them [15].were no significant differences between the previousvalues and those determined at 30 min (1.82 { 1.28 Discordant xenotransplantation, the use of organs

from common animal species for human transplanta-pmol/ml) and 60 min (1.92 { 1 pmol/ml) (P Å 0.59).Then, they went down to values even lower than the tion, could be a solution for this situation. However,

the clinical application of this procedure is not possiblebasal determinations (0.64 { 0.27 pmol/ml) (P Å0.0177) (Fig. 2). nowadays, as far as it results in a hyperacute vascular

rejection. According to this, most of the present re-Constitutive and inducible NO synthases (Fig. 3)were active in the renal parenchyma 30 min after the search in this field is aimed to determine the patho-

FIG. 1. Nitrites (renal vein).


377CASTILLO ET AL.: NO PRODUCTION DURING VASCULAR REJECTION

FIG. 2. Nitrites (renal artery).

physiologic basis of this rejection, a previous and a nec- blood pressure (causing an intense vasodilatation) andthe platelet physiology [18, 19]. On the other hand, theessary way to prevent it and to control it [16].

The analysis of the hyperacute vascular rejection Inducible NO-s is normally absent from the macrophagesand other reticuloendothelial system cells, but once theseleads to the study of the endothelial cell biology, as this

cell represents not only the first and main target of the cells are activated by cytokines, it synthesizes largeamounts of NO with cytotoxic activity [20, 21].procedure, but also, a great part of the mechanisms

leading to its development can be explained as modifi- Once determined the role of NO as a nonspecific im-mune effector, we wondered if this NO could take partcations of normal endothelial cell functions (coagula-

tion, vascular tone, and inflammation controls) [17]. in the development of the hyperacute vascular rejec-tion: the binding of preformed natural antibodies toThe study of the endothelial cell biology has recently

introduced an important new mediator, the nitric oxide their epitopes on the endothelial cell could activate theNO synthase in the graft reticuloendothelial cells, gen-(NO). The biological functions of this NO are clearly re-

lated to the NO-synthase isoform responsible for its pro- erating NO with cytotoxic activity and, in that way,mediating host tissue damage.duction. Constitutive NO-s is always present in endothe-

lial cells, releasing intermittent and small amounts of After an informatized search, we found several ex-perimental studies trying to correlate the productionNO. This NO transmits different signals, regulating the

FIG. 3. Nitric oxide synthases (renal parenchyma; xenotransplantation).



FIG. 4. Nitric oxide synthases (renal parenchyma; autotransplantation).

of cytotoxic NO with the host-versus-graft and graft- cant, and therefore, may represent increases in NOlevels in the blood coming from the graft.versus-host reactions during the course of an immune

response to alloantigens [22]. We also read interesting Our next step tried to investigate if those changesregistered in the nitrite vein levels were really deter-reports about the role of the cytotoxic NO in the devel-

opment of endotoxic shock [23], but we did not find any mined by the rejection (as in our hypothesis) or, onthe contrary, they only were the result of the receptorwork trying to determine a relationship between this

two subjects: NO and hyperacute rejection. organism reaction against the surgical injury of thetransplantation procedure. In order to answer thisNO is a highly unstable, gaseous free radical, with

a half-life of only 3 to 5 sec, before undergoing sponta- question, we determined the time evolution of the plas-matic nitrite levels in the renal artery (as a sample ofneous inactivation by oxidation to nitrite and nitrate

(the stable endproducts of NO metabolism), a reaction systemic blood), assuming that, in such a case, theselevels should increase as the surgical response media-catalyzed by oxygen-derive radicals [24].

Figure 1 represents the ANOVA test for the evolution tors would play their part.Figure 2 represents the ANOVA test for the evolutionof the nitrite plasmatic levels in the renal vein. It shows

that, after the kidney reperfussion, nitrite levels in- of the nitrite plasmatic levels in the renal artery. Afterthe kidney reperfusion, nitrite levels remain un-crease, reaching the maximum level 5 min later, and

then decreasing until the vein thromboses. Both the changed during the next 60 min; then, as the renalartery thromboses starts, their level decreases. Theseinitial increase and the overall evolution are signifi-

FIG. 5. Nitric oxide synthases (renal artery; xenotransplantation).


379CASTILLO ET AL.: NO PRODUCTION DURING VASCULAR REJECTION

FIG. 6. Plasmatic cGMP (renal vein; xenotransplantation).

results, taken together with the previous renal vein into account that in other situations with increased NOproduction there is no increase in cGMP levels [25].values, make a systemic origin for the NO production

very unlikely. Thus, the transplanted organ seems to After the identification of the inducible NO synthaseas being responsible for the production of cytotoxic NObe the place where NO is synthesized.

Going on with a logical development of our working by the graft, the last question we tried to resolve was:Has the NO synthase been induced during the hyper-hypothesis, we tried to answer two new questions: Was

the NO synthesized de novo or was the renal vein plas- acute rejection or is it the result of the organ surgicalhandling and ischemia?matic nitrite level increase the result of a previous or-

ganic deposit? Has the determined NO cytotoxic activity? To answer to this last question we determined theNO-synthases activities in samples of renal paren-In order to resolve both problems we measured the

NO synthase (constitutive and inducible) activity in chyma after renal pig heterothopic autotransplanta-tion. The results are shown in Fig. 4. While the valuestissue samples taken from the renal parenchyma and

the renal artery, after the organ extraction. for the constitutive NO synthase isoform are the samethat in the discordant xenotransplantation cases, thereThe results obtained are shown in Figs. 3 and 5. As

the graphics represent, Inducible NO synthase activity is no inducible NO synthase activity in this group. Asthe only difference between both groups is precisely theis already present in the renal parenchyma samples

obtained 30 min after the organ reperfusion, and its rejection development, we may conclude that cytotoxicNO is released after discordant xenotransplantation,absolute value is greater than the one obtained for the

constitutive NO-s activity. As the inducible isoform re- and it may be involved in the pathogenesis or patho-physiology of hyperacute vascular rejection.leases greater amounts of NO (nanograms) than the

constitutive isoform (picograms), it can be established From a pathophysiologic perspective, the hyperacuterejection could be the result of local vasoconstriction,that the greatest part of the NO released by the graft

has been recently produced. Thus, the plasmatic NO secondary to a decrease in NO production owing to en-dothelial injury. This phenomenon could be associatedlevel increase is not the result of a previous deposit.

For the same reasons, and according to the relationship to the increase of other potent vasoconstriction prod-ucts, like endothelins (personal communication) [26].between the NO biological properties and the NO syn-

thase leading to its release, most of this NO will be However, this is not contradictory with our results, asmost of the NO released by the graft during the hyper-cytotoxic and it will not stimulate the soluble guanylate

cyclase. This, together with the simultaneous release acute rejection is the result of the activation of theinducible NO synthase and for this reason is no longerof Endothelins, could explain why, in hyperacute rejec-

tion, an intense vasoconstriction is noted. a vasodilator but a cytotoxic molecule.Even though we have been trying to describe and toThe difference between both enzymes activity mea-

sured in the renal parenchyma and the renal artery explain our results in a logical way, we cannot forgetwe are dealing with a difficult problem. Many biologicalcan be explained because the quantitative difference

in the rejection rate in both organs, due to the different systems are activated and multiple cytokines are beingreleased. It is difficult to determine which is the causenumber of endothelial cells, and mainly, of the reticulo-

endothelial cells that they contain. and which is the result of the mediators.The decrease in blood flow to a hyperacutely rejectingThe lack of elevation of the cGMP levels in the blood

obtained from the renal vein is not surprising, taking organ could lead to sequestration of macrophages



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nitric oxide production during the hyperacute vascular rejection

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