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From the Department of Nephrology,*
University of Essen,
Essen, Germany; and the Semmelweis
Medical University,
Budapest, Hungary
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, interleukin (IL)-1,
IL-6, inducible nitric oxide synthase (iNOS),
and interferon (IFN)-
messenger RNA (mRNA)). In controls,
renal ischemia of 30 minutes was nonlethal, whereas 73% of the
animals died within 48 ± 18 hours, after 45 minutes of
ischemia. All different doses of LPS protected the animals from lethal
renal ischemia/reperfusion injury. Starting at similar levels,
serum creatinine increased significantly in controls but not in
LPS-pretreated animals over time. As early as 2 hours after
reperfusion, tubular cell damage was significantly more
pronounced in controls than in LPS-treated mice. In controls,
tubules deteriorated progressively until 8 hours of reperfusion.
At this time, more than 50% of tubular cells were destroyed.
This destruction was accompanied by a pronounced leukocytic
infiltration, predominantly by macrophages. In
contrast, LPS pretreatment prevented the destruction of kidney
tissue and infiltration by leukocytes. The terminal
deoxynucleotidyltransferase-mediated UTP end-labeling assay revealed
significantly more apoptotic cells in controls compared with
LPS-pretreated animals. IL-1, IFN-, and iNOS
mRNA expression did not differ between the groups throughout the time
points examined. However, the expression of TNF- mRNA was
significantly increased at 2 hours and IL-6 mRNA was significantly
down-regulated before ischemia and shortly after reperfusion in the
LPS-pretreated kidneys. Therefore, we found that sublethal
doses of LPS induced cross-tolerance to renal ischemia/reperfusion
injury. Our data suggest that increased TNF- and reduced IL-6 mRNA
expression might be responsible. However, more studies are
needed to decipher the exact mechanism.
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Introduction |
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50%.5
The pathophysiological mechanisms leading to acute ischemic renal
failure are not completely understood. There is no clinically effective
therapy that prevents ischemic injury completely.6
Moreover, this so called ischemia/reperfusion injury contributes to
renal damage in transplantation,7
revascularization
procedures, and periods of hypoperfusion.8 Severe reduction of renal blood flow causes cell damage by high-energy phosphate depletion and the subsequent failure to maintain physiological ion gradients across the cellular membrane. The severity of the injury depends on the duration of ischemia and the availability of collateral perfusion, although, paradoxically, the restoration of blood flow itself is associated with further tissue damage. Reperfusion with oxygenated blood is associated with the generation of free radicals and thus lipid peroxidation, polysaccharide depolymerization, and desoxyribonucleotide degradation. Injured endothelial cells fail to initiate the relaxation of vascular smooth muscle cells, release potent vasoconstrictors, and swell; the permeability is increased, and finally, leukocytes and platelets are trapped and accumulate in the microcirculation and the tissue. Eventually this results in a progressive loss of perfusion and further tissue damage.7,8
Both sepsis and endotoxemia are generally regarded as destructive processes.9-12 Preconditioning with endotoxin results in adaptation or tolerance, which is characterized by a reduced systemic response to a subsequent challenge with a large dose of homologous or heterologous endotoxin.9-12 Although endotoxin has been used to induce resistance against a subsequent identical insult,13 it has also been demonstrated that endotoxin provokes cross-tolerance against other forms of injury. Some authors reported endotoxin-derived protection against ischemia/reperfusion injury in myocardium14 and liver.15
In this study, we established a new model for endotoxin-induced cross-tolerance to renal ischemia/reperfusion injury. To gain some insights into the underlying processes, we evaluated cellular infiltration and cytokine production in a second set of experiments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male CD57/Bl mice (weight, 20–30 g) were used as experimental animals, maintained on a standard diet, and given water ad libitum. The animal protocol was reviewed and approved by a governmental animal care and research committee.
Experimental Design and Operation
Operative procedures were performed under general anesthesia induced by 5.3 mg/100 g nembutal and 0.02 mg/100 g atropin-sulfate administrated intraperitoneally. After a midline laparatomy incision, renal artery and vein of the left kidney were isolated and occluded with a clamp. After ischemia, the clamp was withdrawn, the right kidney was removed, the laparatomy incision was closed, and the animals were allowed to wake up.
In the first step, lethal renal ischemic time was determined in the mice. Two different times of ischemia were examined: 30 minutes and 45 minutes (n = 26/group).
In the second step, we established a model for endotoxin-induced
cross-tolerance to lethal renal ischemia/reperfusion injury. Animals
were treated according to three different protocols for
lipopolysaccharide (LPS) administration (Escherichia
coli, serotype 0111:B4; Sigma Chemical Co., St. Louis, MO) (Table 1)
(n = 12/group).
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Functional Parameters
Serum creatinine concentrations were determined photometrically with a commercially available test kit (Boehringer Ingelheim, Ingelheim, Germany).
Histological Analysis
Paraffin sections of kidneys fixed in 4% neutral buffered formalin were stained with hematoxylin and eosin and periodic acid-Schiff reagent. Samples were coded and examined in a blinded fashion. Tubular damage and leukocyte infiltration were semiquantitatively evaluated on a scale from 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe). Additionally, neutrophils were manually counted as cells per field of view at x400 magnification.
Immunohistological Analysis
Fresh frozen sections were stained with antibodies against lymphocytes (CD4 and RM45, Pharmingen, San Diego, CA) and macrophages (CD11b, M1/70, Pharmingen) to establish the involvement lymphocytes. Furthermore, we evaluated apoptosis with the TUNEL assay based on the description of the manufacturer (Boehringer Mannheim, Mannheim, Germany).
Positively stained cells were counted at x400 magnification and described as cells per field of view.
Reverse Transcriptase-Polymerase Chain Reaction
Total RNA Isolation
Total RNA was extracted and used for reverse transcriptase-polymerase chain reaction (RT-PCR). A part of the kidney was stored in 500 µl of cold lysis solution, containing 4 mol/L guanidine isothiocyanate (Sigma), 25 mmol/L sodium citrate (pH 7.0), 0.1 mol/L ß-mercaptoethanol, and 0.5% sarcosyl, and frozen in liquid nitrogen. Total RNA was extracted by the modified guanidine-isothiocyanate preparation method.16 Briefly described, frozen tissues were mixed with 4 ml guanidine isothiocyanate buffer (4 mol/L guanidine isothiocyanate; Sigma) and acid phenol-chloroform (pH 4; Sigma), and homogenized. The samples were centrifuged at 1500 g for 10 minutes at 20°C. The supernatant was treated with an equal volume of isopropanol. The mixture was centrifuged, and the RNA was washed with RNeasy Total RNA Isolations Kit (Qiagen GmbH, Germany) and stored at -80°C until further processing. RNA concentration was measured spectrophotometrically.
Reverse Transcription
RNA was amplified by reverse transcription (RT) with an oligo(dT)12–18 primer (Life Technologies, Inc., Grand Island, NY). Total RNA (1 µg) was added to 0.5 µg of primer. A reaction mixture was added containing buffer solution (50 mmol/L Tris-hydrochloride buffer, pH 8.3, 75 mmol/L potassium chloride, 5 mmol/L magnesium dichloride, 5 mmol/L dithiothreitol; Life Technologies, Inc.); 1 mmol/L each of adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, and cytosine triphosphate (deoxynucleoside triphosphates from Boehringer Mannheim GmbH); 40 U/µl of recombinant ribonuclease inhibitor (Promega), and 0.5 µl of 200-U/µl Maloney-murine leukemia virus reverse transcriptase (Life Technologies), and the first chain reaction was allowed to proceed (36°C, 1 h). The reaction was halted by heating to 95°C for 5 minutes followed by cooling on ice.
Amplification of Specific Complementary DNA
Specific complementary-DNA products corresponding to mRNA for
TNF-, IFN-, IL-1, IL-6, inducible nitric oxide synthase
(iNOS), and ß-actin17
were amplified by PCR. A
1-µl sample was taken from the RT reaction for PCR, which was
performed in PCR buffer (750 mmol/L Tris-hydrochloride, pH 9.0, 200
mmol/L
(NH4)2SO4,
0.1% (w/v) Tween, 20 mmol/L magnesium dichloride; Dianova, Hamburg,
Germany), using 0.2 mmol/L of each deoxynucleoside triphosphate, 1
µmol/L of both primers (Eurogentec, Belgium), and 2.5 U Thermus
aquaticus (Taq) DNA polymerase (Dianova). A
Perkin-Elmer Thermal Cycler (Model 2400, Perkin-Elmer, Norwalk, CT) was
used for amplification with the following sequence profile: initial
denaturation at 94°C for 3 minutes followed by 30–40 cycles of
three-temperature PCR (denaturing, 94°C for 30 seconds; annealing,
55°C for 30 seconds; extension, 72°C for 30 seconds) and ending
with a final extension at 72°C for 7 minutes and cooling to 4°C.
Gel Electrophoresis
The amplified PCR product was identified by electrophoresis of 10-µl sample aliquots on 1.5% agarose gel stained with 0.5 µg/ml of ethidium bromide. The sample products were visualized by UV transillumination, and the gel was photographed. Specific products were identified by size in relation to a known 1-kb oligonucleotide DNA ladder (Life Technologies) run with each gel. Cytokine complementary DNA was semiquantitated by densitometric comparison with ß-actin (internal control) from the same sample after the positive image was digitized by video for computerized densitometry. The results are given as the ratio of intensity of cytokines and ß-actin mRNA ± SEM.
Statistical Analysis
Data were expressed as mean ± SEM. Parametric data were compared using Student’s t-test. Nonparametric data were tested using Mann-Whitney analysis of ranks. P < 0.05 was accepted as statistically significant.
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Results |
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After 30 minutes of renal ischemia, all animals
(n = 26) survived until the end of the follow-up
(15 days). After 45 minutes of renal ischemia (n
= 21), 20 animals died after 48 ± 24 hours (74%). One
animal survived for 13 days (Figure 1)
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After pretreatment with LPS, all animals (n = 35) survived for more than 50 days, independent of the administration protocol. Therefore, 45 minutes of ischemia was chosen for the following experiments.
Whereas the body weight of vehicle-treated animals remained constant during the period observed, it decreased in LPS-treated mice after the first administration by approximately 10% and recovered thereafter.
Serum creatinine was similar before and 15 minutes after perfusion in
both groups (controls: 1.2 ± 0.06 and 0.76 ± 0.07;
versus LPS: 0.94 ± 0.11 and 0.65 ± 0.06 mg/dl).
One hour after ischemia, creatinine had increased in both groups
(controls: 1.69 ± 0.19; versus LPS: 1.11 ± 0.17
mg/dl). However, this increase was more pronounced in controls.
Thereafter creatinine remained constant in the LPS group (1.21 ±
0.16 mg/dl), whereas it increased in controls (3.03 ± 0.18
mg/dl). One hour after ischemia, the differences between the groups
reached statistical significance (Figure 2)
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Immediately before ischemia, minor tubular damage was observed in
the LPS group, although kidneys from controls had no apparent
morphological changes. At 15 minutes after reperfusion, tubular damage
was similar in LPS- and vehicle-treated animals. However, the damage
progressed slowly in LPS-pretreated animals up to 2 hours after
reperfusion and returned to a normal appearance after 8 hours. The
degree of leukocyte infiltration was low in these animals. Kidneys of
vehicle-treated animals deteriorated dramatically over time with an
accompanying strong infiltration of leukocytes. The infiltrate
consisted predominantly of neutrophils. By 8 hours after reperfusion,
almost half of the kidney was destroyed in these animals (Table 2
and Figure 3
).
Furthermore, we observed more cells undergoing apoptosis in controls
than in LPS-pretreated animals. As revealed by the TUNEL assay,
1.80 ± 0.64 cells per field of view stained positive in
LPS-pretreated animals compared with 0.53 ± 0.17 in controls at
16 h after ischemia (P < 0.01).
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At 2 hours, the expression of TNF- mRNA was significantly
higher in the LPS group than in controls. Furthermore, in LPS-treated
animals, macrophage-associated IL-6 mRNA levels were threefold lower
before ischemia than in controls and remained significantly lower at 15
minutes after reperfusion, but these levels increased to higher levels
than in controls thereafter. On the other hand, in both groups, IL-1
was up-regulated at 15 minutes, low until 2 hours, and up-regulated at
8 and 16 hours. The mRNA patterns of IFN-, IL-1, and iNOS did not
differ between the groups (Figure 5)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite experiments, the underlying mechanisms and cellular mediators
responsible for the endotoxin-related tolerance to ischemia/reperfusion
injury remain elusive. It is known that polymorphonuclear cells are
involved in this process. The results of in vitro and
in vivo experiments have supported the conclusion that
macrophage-associated cytokines, such as TNF-, IL-1, IL-6, and
IFN-, may play a major role in this process.19
The most
widely examined cytokine in the development of endotoxin tolerance is
TNF-. We demonstrated an increased level of TNF- mRNA expression
after renal ischemia in LPS-pretreated animals. However, despite an
increased TNF- mRNA level, renal injury was reduced. These results
indicate an involvement of TNF- and particularly a missing response
to TNF-. It is known that TNF- appears almost immediately on LPS
injection in mice. However, repeated injections of LPS resulted in
unresponsiveness, and no further TNF- production was detectable for
at least days.7
In contrast to our experiment, these
results indicated a direct involvement to TNF- because the level of
TNF- correlated to LPS unresponsiveness.
However, the TNF- level also increased in another experiment after
endotoxin pretreatment and hepatic ischemia/reperfusion injury, and
again this elevation correlated to a protection against lung injury
after hepatic reperfusion.15
The mechanism for the
enhanced production of TNF- in animals treated with LPS and
subsequently subjected to ischemia is difficult to explain. The most
likely hypothesis for the protective effects induced by nonlethal LPS
administration is a transient induction of TNF-. This early increase
of TNF- will then provide protection against the subsequent organ
damage by a down-regulation of TNF receptors.20
Otherwise
it is hard to explain why the damages were reduced by LPS pretreatment
despite a higher TNF- mRNA expression.
In parallel to these results, we were not able to detect any
differences in the kinetics of IL-1 and IFN- mRNA expression between
the groups. However, the level of IL-6 mRNA was significantly reduced
before ischemia and shortly after reperfusion in LPS-pretreated
animals. In an in vivo study, Hewitt et al demonstrated that
intermittent ischemia (preconditioning) resulted in significantly
decreased IL-6 and TNF- expression as compared with continuous
ischemia (without conditioning).22
In addition, it
has been reported that daily injections of LPS in rats resulted in a
measurable release of TNF- and IL-6 within the first few days only
and was abrogated thereafter.22
Our data indicate that
repeated treatment of endotoxin can inhibit IL-6 mRNA expression in the
injured organ. We think that reduced IL-6 expression may reflect a
state of unresponsiveness of macrophages. This may be related to
down-regulation of the TNF receptors. On the other hand, IL-6 by itself
is a strong chemoattractant and may lead to the infiltration and
consequential activation of leukocytes. This hypothesis would explain
the reduced infiltration observed in the LPS group. The reduced
infiltration by these leukocytes could further explain the lower degree
of damage and apoptosis in these animals.
However, another hypothesis can be postulated. Hypoxia is one of the most pronounced promoters of necrosis/apoptosis by itself. As a result of necrosis/apoptosis, leukocytes infiltrate to phagocytose the damaged tissue. If LPS pretreatment reduces the susceptibility of tissue cells to hypoxia-induced cell death per se, the degree of damaged tissue would be reduced, and fewer leukocytes would infiltrate the affected organs.
Cytokines induce iNOS gene expression23 and thus increase NO levels. After ischemia and reperfusion, injured vascular cells and adherent leukocytes produce free radicals and inactivate NO, which may promote vasoconstriction and increase permeability, local edema, and leukocyte adhesion. Because we did not find any differences in iNOS mRNA expression between our groups, we would conclude that NOS is of minor importance for ischemia/reperfusion in our model. However, we examined mRNA expression of cytokines only in organs and not in the blood or specifically in the endothelium. Thus, it is possible that differences related to certain cells, eg, endothelial cells, were not detected. Additionally, we used RT-PCR to detect the mRNA levels of cytokines, while others examined the protein level in the blood or, for endotoxin tolerance, in the peritoneal macrophages. Whole-blood assays are certainly reasonable in septicemia, but, based on the short half-life of the mediators involved, these assays seem to poorly reflect processes in a single organ such as the kidney. Therefore we did not perform such assays. Furthermore, although peritoneal macrophages are undoubtedly a good model for LPS-tolerance, their function and relation to renal injury are unknown and hard to predict. Additionally, most experiments were performed in vitro and not in vivo.
How LPS pretreatment confers protection against renal ischemia/reperfusion injury remained unclear but is undoubtedly multifactorial. Early investigators believed that low-dose LPS stimulates the reticulo-endothelial system such that subsequent larger doses of LPS are more actively cleared.24 More recently, the predominant mechanism emphasized is that low-dose LPS alters monocyte secretion of a variety of inflammatory mediators including proteolytic enzymes, arachidonic acid metabolites, reactive oxygen species, and cytokines.25-30 On the other hand, low-dose LPS could directly down-regulate the expression of endothelial-cell adhesion receptors, which could account for the decrease in tissue sequestration of neutrophils.31 Key molecules involved in this endothelial cell-neutrophil adhesion are intracellular adhesion molecule-132 and E-selectin.33,34 Both are an ideal molecular indicator of endothelial cell activation in response to ischemia/reperfusion injury. In in vitro models, hypoxia/reoxygenation stimulated endothelial cells to express adhesion molecules and increased adhesion of neutrophils to the endothelial surface.35 Neutrophil depletion has been shown to protect rats against ischemic renal injury in some studies.36,37 Thus, given the complexity of events preceding neutrophil invasion into the tissues, one could postulate that LPS pretreatment may inhibit the activation of endothelial cells.
Although renal failure after ischemia and reperfusion can be managed by
dialysis, such injury influences the outcome of transplantation,
revascularization procedures, and episodes of hypoperfusion. In this
experiment we developed a new model for endotoxin-induced
cross-tolerance to renal ischemia/reperfusion injury. Although this
model may seem quite extreme, similar damages may occur in the clinical
setting for non-heart-beating donors. TNF- and IL-6 may play
a central role in this process. However, future studies are needed to
decipher the exact causes of the increased survival in this model.
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Footnotes |
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Accepted for publication September 4, 1999.
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-1-protease inhibitor: cooperative use of lysosomal proteases and oxygen metabolites. J Clin Invest 1984, 73:1297-1304
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, LPS
group; 
, controls; *P <
0.05.