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Journal of Investigative Surgery, 14:1–8, 2001
Copyright c2001 Taylor & Francis
0894-1939/01 $12.00 + .00
Original Research
Exogenous Nitric Oxide Protects Kidney
from Ischemia/Reperfusion
Enrique S ´anchez-P ´erez-Verd´ıa,
MD,
Fernando L ´opez-Neblina,
MD, MSc,
Eliseo Portilla, DVM, MSc,
Genaro Ort´ız, MD, MSc,
Alejandro Gonz ´alez-Ojeda,
MD,
and Rigoberto Alvares, MD
Surgical Research Division, Centro
de Investigaci ´on Biom ´edica de
Occidente, Instituto Mexicano del
Seguro Social, and Colegio de
Especialistas en Cirug´ıa General
del Estado de Jalisco A.C.,
Guadalajara, Jalisco, Mexico
ABSTRACT Blockade of NO production is followed by an increase in
leukocyte rolling and adhesion resulting in some deleterious effects of
ischemia. Preischemic administration of NO protects vascular integrity
after reperfusion. Exogenous NO causes a direct reduction in leuko-
cyte adhesion. This work was performed to test the hypothesis that
exogenous NO administered during the preischemic period to the kid-
ney alone, without coming into contact with the leukocytes, could also
reduce leukocyte–endothelium adhesion. Adult rats were subjected to
in situ isolation of the left kidney. Solutions were infused through the
renal artery and drained through an incision in the renal vein, thus avoid-
ing the systemic circulation. Group IC rats served as an ischemic control,
and received 0.9% saline. Group NP received Na nitroprusside. Group S
was a nonischemic control. Groups IC and NP were subjected to 75 min
of renal ischemia. After this period, vascular structures were repaired and
reperfusion allowed. A right nephrectomy was performed. Serum urea
and creatinine, myeloperoxidase activity, and histopathological stud-
ies were carried out at different intervals after reperfusion. Survival at
15 days was 46%, 80%, and 100% in groups IC, NP, and S, respec-
tively. Differences between groups for serum urea and creatinine were
significant only during the first seven days. Myeloperoxidase values were
significantly higher in group IC. All rats from group IC and only 20%
from group NP showed histological evidence of necrosis. Thus, exoge-
nous NO is protective and acts selectively upon the kidney, modulating
its interactions with polymorphonuclear cells after ischemia/reperfusion.
KEYWORDS adhesion molecules, endothelium, ischemia/reperfusion, kidney,
neutrophil infiltration, nitric oxide, rat
The authors thank Adolfo C ´
ardenas,
Ernesto Duarte, David Garc´
ıa, Alberto
Ramos, Sergio Rodr´
ıguez, and
Gabriela Santill´
an for their kind
assistance in this study.
Address correspondence to Dr. Eliseo
Portilla, CIBO, Surgical Research
Division, AP 1-3838, Guadalajara, Jal.,
44340 Mexico. E-mail: eportilla@mail.
udg.mx
1
Original Research
Nitric oxide (NO) has been identified as an
important molecular effector in a variety of
physiological and pathological conditions.
The role of NO in ischemia and reperfusion (I/R) is
still controversial. Some authors state that NO may
be deleterious after I/R due to its interaction with
oxygen-based free radicals upon reperfusion [1–3].
At the same time, others have found that when en-
dogenous NO production is blocked by antagonists
such as nitroG-methyl ester-L-arginine (L-NAME) or
NG-monomethyl-L-arginine (L-NMA), the deleteri-
ous effects of I/R, including leukocyte rolling and
adhesion in the postcapillary venules, are increased
[4–6]. In the same sense, exogenous NO donors and
endogenous NO have been shown to exert a protec-
tive effect from I/R and endotoxemic damage, by
maintaining vascular integrity [7, 8]. Some attribute
the protective effects of NO to its potent action
as a free-radical scavenger [9] and its ability to main-
tain the microvascular integrity as an endothelium-
derived relaxing factor [10]. It has been demons-
trated that NO decreases platelet adhesion, and that
this action is related to the inactivation of superoxide
anions [4, 11].
Furthermore, the results of recent research have
proposed that the principal beneficial effect of NO is
exerted by the alteration of inflammatory responses
through the modulation of interactions between
leukocytes and endothelial cells (ECs). It has been
shown, by previous treatment with exogenous NO
at different intervals before reperfusion, that there
is a decreased neutrophil infiltration into the reper-
fused zone and an alteration to the rolling, acti-
vation, and adhesion of polymorphonuclear cells
(PMNs) [12, 14]. The protective effect observed in
Q1
these studies could be due to an interaction between
NO and inflammatory cells, because NO donors
were administered to the systemic circulation after
ligation of the renal pedicle. In this technique, the
treatment did not have any effect on the ischemic
endothelium and most probably the locus of action
was on the circulating leukocytes. However, it is not
clear if exogenous NO exerts the same effect when
administered selectively and exclusively to the en-
dothelium of the preischemic kidney, avoiding con-
tact with leukocytes from the general circulation.
Therefore, the aim of the present study was to ex-
amine specifically the role of exogenous NO donors
on the inflammatory response observed after reper-
fusion. Exogenous NO donors were administered
exclusively to the kidney, thereby avoiding contact
with the systemic circulation.
METHODS
All experiments were carried out following insti-
tutional guidelines for the use and care of labora-
tory animals. In total, 105 Sprague-Dawley male rats
(250–300 g) from our local animal facility were
subjected to 75 min of warm ischemia by clamp-
ing the vessels of the left renal pedicle. Briefly, un-
der intraperitoneal sodium pentobarbital anesthesia
(60 mg/kg body weight) and through a midline la-
parotomy, the abdominal viscera were retracted to
the right side. Under an operating microscope, the
left renal hilus was dissected with ligation and divi-
sion of the adrenal and gonadal arteries and veins.
The perirenal fat was preserved. The renal vascular
pedicle was occluded temporarily using a microvas-
cular bulldog clamp on the renal artery near the
aorta, and an occluding, untied 6-0 silk suture on
the renal vein near the vena cava.
Immediately after this, a longitudinal draining
venotomy was performed on the renal vein between
the kidney and the silk, and for renal perfusion the
renal artery was cannulated with a 27-gauge needle
connected by an extension to a syringe. Thus, an
isolated perfusion circuit to the organ was created
by draining the solution through the venotomy and
removing it from the abdominal cavity with a pneu-
matic aspirator, thereby avoiding access to the sys-
temic circulation (Figure 1). This technique has been
tested and standardized in a previous study [15]. The
animals were divided into three groups. Group IC,
an ischemic control group (n=35), received 2 mL
of 0.9% saline solution infused over 1 min. Group
NP, an experimental group (n=35) with Na nitro-
prusside (Na-NP) as an exogenous NO donor, re-
ceived an infusion of 2 mL of dilute Na-NP (1.5 mg
Na- NP in 10 mL of 0.9% saline solution mixed im-
mediately before administration with 0.2 mL hep-
arinized autologous fresh blood). Addition of the
2 E. S ´
ANCHEZ-P ´
EREZ-VERD´
IA ET AL.
Q1: Au: Please cite ref 13.
Original Research
FIGURE 1 Model of exclusive in situ renal perfusion and
study protocol. Immediately after induction of warm ischemia
in the left kidney, cannulation of the renal artery and veno-
tomy were performed in order to flush the kidney with a so-
lution containing the drug, and to avoid its passage to the
systemic circulation. Reperfusion to the left kidney took place
after 75 min with the renal vessels repaired, followed by a right
nephrectomy.
autologous blood promotes the liberation of NO
from the nitroprusside molecules when these come
into contact with sulfhydryl groups on the erythro-
cyte membrane [16]. Three minutes were allowed
after infusion of the drug (sufficient time for NO
liberation and diffusion to the endothelium) and
a new perfusion with pure saline solution was per-
formed to wash out Na-NP metabolites. Group S was
a sham group (n=35). These animals were subjected
to the same procedure of in situ isolated renal perfu-
sion with physiologic solution only, but without any
period of ischemia.
Immediately after the end of perfusion, the renal
vessels were repaired with an uninterrupted suture
of 10-0 nylon in the vein and a single stitch point in
the puncture hole of the artery. The abdominal con-
tents were repositioned in the abdominal cavity, the
incision was covered with a moistened polyethylene
sheet, and 75 min of total ischemic time was allowed.
At the end of the ischemic period, the abdominal
cavity was reopened and the silk in the vein and the
clamp in the artery were removed allowing reper-
fusion of the kidney. In group S, reperfusion took
place immediately after renal vessel reconstruction,
thereby causing a total ischemic time of approxi-
mately 15 min for this group.
After achieving hemostasis and confirming an ade-
quate reperfusion of the kidney, a right nephrectomy
was performed. The abdominal wall was closed with
2-0 silk suture.
Animals were fed a standard pellet diet (Ralston
Rations, Kansas City, KS) and had free access to wa-
ter while they were observed for survival period of
15 days. Laboratory studies (n=10 per group) in-
cluded serum urea (SUr) and serum creatinine (SCr),
which were assessed at 24, 48, and 72 h and at 7 and
15 days. For these tests, 1 mL central venous blood
was removed and replaced with the same volume
of lactated Ringer’s solution. SUr and SCr values
were determined with colorimetric tests (diacetyl- Q2
thiosemicarbazide technique for SUr; Jaffe method
for SCr) in a Coleman Jr. II spectrophotometer
(Perkin Elmer Co, Oak Brook, IL).
Neutrophil infiltration was estimated in five ani-
mals from each group by measuring the activity of
myeloperoxidase (MPO) in the renal tissue. MPO
is a heme-containing enzyme found primarily in
PMNs, and can be used as an indirect estimator of
neutrophil infiltration into the tissue. This was mea-
sured in accordance with the method described by
Bradley and Prietbat [17]. After a 2-h period of reper-
fusion, the abdominal aorta was cannulated and the
kidney flushed with cold saline solution (4◦C) and
immediately frozen and stored in liquid nitrogen
until the assay was performed. Kidney tissue sam-
ples were weighed and homogenized in 10 mL of
an aqueous solution B containing 0.021% K2PO4,
0.663% KH2PO4, and 0.5% hexadecyltrimethyl am-
monium bromide (HTBA; all obtained from Sigma
Chemical Co., St. Louis, MO). The homogenates
were frozen–thawed 3 times in order to induce cellu-
lar lysis and then centrifuged at 2000 rpm for 10 min
at 0◦C. After this, 0.1 mL supernatant was added to
2.9 mL freshly prepared solution C (described later)
and assayed for MPO spectrophotometrically at a
fixed wavelength of 460 nm (Sequoia-Turner Co.
model 690). The change in absorbance was mon-
itored every minute for 10 min. Solution C con-
tained 0.0105 g K2HPO4and 0.3315 g KH2PO2in
40 mL distilled water, to which was added 5 mL of
EXOGENOUS NO AND I/R 3
Q2: Au: Ok acetyl?
Original Research
0.017% solution of dianisidine in methanol and
5 mL of 0.006% hydrogen peroxide in distilled water.
One unit of MPO was defined as the amount that
degraded 1 mmol peroxide/min at 25◦C. The unit
value per gram of tissue was calculated using the fol-
lowing formula: (highest absorbance/10 min)/g tis-
sue used/0.0113 =MPO/g tissue, where 0.0113 is a
constant [18].
Five additional animals per group were used for
histological studies. Using light microscopy, sam-
ples of the kidney obtained 24 h after ischemia/
reperfusion were evaluated for acute tubular necro-
sis, vesicular degeneration, and hyperchromasia in
the proximal convoluted tubules, according to pre-
viously described criteria [19]. The evaluation of his-
tological damage was carried out in a blind study.
Statistical analysis included analysis of variance
(ANOVA), with Fvalues considered significant at
an alpha value of .05 for parametric variables (SUr,
SCr, and MPO activity). Survival was plotted by
Kaplan–Meier curves, and the final survival at
15 days was analyzed using Fisher’s exact test with
Yates’s correction.
Q3
RESULTS
Survival
Figure 2 shows differences in survival curves be-
tween the three groups. Final survival at 15 days was
FIGURE 2 Survival curves. There was a statistically signifi-
cant difference in survival at 15 days only between groups IC
andS(p<.05).
46% in group IC, 80% in group NP, and 100% in
group S. There were statistically significant differ-
ences in survival between the groups IC and S from
day8to15(p<.001). No statistically significant
difference was found between groups NP and S ( p=
.22). Although group NP showed a better survival
than group IC, this did not attain statistical signifi-
cance ( p=.10).
Renal Function Test
Figure 3 illustrates the changing levels of serum
urea (SUr) at 24, 48, and 72 h and at 7 and 15 days
for each group. It can be observed that during the
first 24 h, no statistically significant differences ex-
isted between groups IC and NP, but they did exist at
this stage when compared with group S. Group NP
showed significantly lower levels than group IC at
48 h, 72 h, and at 7 days after the procedure. No sig-
nificant differences existed between groups NP and S
after 48 h. Measurements on group S remained uni-
form and without significant differences from pre-
viously standardized levels of healthy, normal rats
from our animal house. The results in the serum crea-
tinine levels (SCr) were similar to those described for
SUr, with statistically significant differences ( p<.01)
between groups IC and NP at 72 h and 7 days
postreperfusion, but no difference between groups
NP and S (Figure 4).
FIGURE 3 Serum urea levels (SUr). Significant differences
were found comparing the groups as follows: group IC vs.
group S, ∗p<.001; group IC vs. standard control, ∗∗p<.01;
group IC vs. group NP, •p<.05; group NP vs. group S, •• p<
.01. Group S maintained equal levels of serum urea compared
to levels observed in the standard control animals.
4 E. S ´
ANCHEZ-P ´
EREZ-VERD´
IA ET AL.
Q3: Au: Is ok?
Original Research
FIGURE 4 Serum creatinine levels (SCr). Statistically signif-
icant differences were observed between the groups IC and S
at 24 h with ∗p<.05, and between the groups IC and NP with
∗∗p<.05. No statistically significant differences were observed
at any time between groups NP and S.
Neutrophil Infiltration
The values for MPO activity in renal tissue, as an
indirect marker of neutrophil infiltration, are shown
in Figure 5. These correlated well with survival and
renal function. The levels found in group IC were sig-
nificantly higher when compared with both groups
NP and S. No significant differences existed between
these latter two groups. Furthermore, there was no
difference between these groups and previously stan-
dardized levels in similar rats that had not undergone
any kind of procedure (Figure 5).
FIGURE 5 Myeloperoxidase (MPO) activity in renal tissue
as a marker of neutrophil infiltration. In group IC, the levels of
MPO were significantly higher, ∗p<.05, compared to all other
groups. No differences were found between groups NP and S,
or between these and previously standardized levels in similar
healthy normal rats from our laboratory (standard control).
Histology
No significant changes were observed in group S,
and the percentage of animals with changes was
similar to that from kidneys of normal healthy rats
FIGURE 6 In the proximal convoluted tubules, (a) group IC
shows necrosis, nuclear hyperchromasia, and cloudy swelling
with vesicular degeneration of cells resulting in loss of the
cytoarchitecture at the luminal border of the tubule. The
glomeruli exhibit alterations resulting in a loss of integrity in
Bowman’s capsule. (b) Group NP shows 30–40% of protec-
tion, as revealed by reduced necrosis and vesicular degenera-
tion in the proximal convoluted tubule. (c) Neither group S nor
standard control samples (not shown) showed any significant
alterations.
EXOGENOUS NO AND I/R 5
Q4: Au: Please explain art asterisks, in legend.
Original Research
TABLE 1 Histopathological effects on kidney following intra-arterial administration of exogenous nitric oxide donor during
warm renal ischemia (% of rats with morphologically significant change)
Vesicular degeneration of convoluted
Group Hyperchromasia Necrosis proximal tubules
Standard control 0 0 0
Group IC 100 80 100
(ischemic control)
Group NP 60 40 60
(ischemia +Na-NP)
Group S 0 0 20
(sham)
from our laboratory. In group IC, necrosis was
present in all three segments of the proximal con-
voluted tubules in four of five samples, and all ex-
hibited hyperchromasia and vesicular degeneration.
In group NP, necrosis was observed in only 20%
of animals, whereas 60% exhibited hyperchroma-
sia and vesicular degeneration (see Figure 6 and
Table 1).
DISCUSSION
Leukocyte–endothelial cell interaction is a cru-
cial event in I/R injury [20–25]. The decrease in
NO and prostacyclin release, and enhanced produc-
tion of pro-inflammatory substances (oxygen free
radicals, endothelins, lipid mediators, complement
systems and cytokines) promote PMN recruitment
and adherence to the injured area during reperfusion
[26–30].
Leukocyte recruitment is determined by a series
of mechanisms described as activation, rolling, adhe-
sion, and migration [31]. Each stage is characterized
by the expression of different kinds of molecules at
the surfaces of the leukocyte and EC, namely, inte-
grins in the former and the immunoglobulin super-
family in the latter [28, 32]. Many proinflammatory
mediators have the ability to initiate and maintain
this process. However, much less is known about the
endogenous mediators that could reverse or prevent
this event. NO may be an antiadherent and/or anti-
inflammatory molecule.
In previous studies, exogenous NO administered
systemically ameliorated neutrophil infiltration and
showed a protective effect against I/R injury [12, 33].
Studies using intravital microscopy in a mesenteric
I/R model in the rat showed that the systemic
administration of NO reduced PMN rolling and ad-
herence in postcapillary venules [13].
The model used in this work allows us to differen-
tiate the systemic actions of NO from those on the
kidney alone. We changed the administration route
to an in situ isolated renal perfusion, and adminis-
tered an NO donor through the renal artery with the
aim of obtaining a selective effect on the ischemic
kidney and its endothelium. Our hypothesis was that
exogenous NO would modify leukocyte–EC inter-
actions. In this model, circulating leukocytes did not
have contact with the exogenous NO. The significant
reduction of PMN infiltration into the reperfused
kidney, and the decreased mortality with improve-
ment in renal function, can be interpreted as a re-
duction in nephron damage. This was corroborated
by light microscopy.
The I/R protective effect observed in our model
cannot be due to the vasodilator activity of Na nitro-
prusside, because this action disappears after 5 min
of administration [17]. It was administered 75 min
before reperfusion took place, so it can be assumed
that any vasodilator effect was already absent. As
the endothelial cell is the major component for
neutrophil–endothelium interaction in the organ,
we believe that the beneficial action of exogenous
NO could be due to inhibitory signals left in the EC,
which can in some way block the expression of ad-
hesion molecules following the period of ischemia.
This is supported by the demonstration by Lefer that
NO blocks the expression of ICAM-1 in endothelial
cell cultures [34].
Although it can be speculated that exogenous
NO acts over the endothelium, modulating its inter-
actions with PMN after ischemia/reperfusion, fur-
ther molecular studies are necessary to determine
6 E. S ´
ANCHEZ-P ´
EREZ-VERD´
IA ET AL.
Original Research
precisely the level of action by which NO is able
to reduce EC activation and dysfunction.
NO could be used to pretreat organs upon is-
chemia, thereby reducing reperfusion injury. Further-
more, secondary and undesirable effects following
systemic administration of NO, as well as its hy-
potensive effect, can be avoided.
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EREZ-VERD´
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