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and Nitric Oxide in Endotoxemia
*
Nephrology and Hypertension Section, Veterans Administration Medical
Center, Minneapolis, Minnesota.
Department of Veterinary Pathobiology, University of Minnesota,
Minneapolis, Minnesota.
Correspondence to Dr. Edgar A. Jaimes, Nephrology and Hypertension Section IIIJ, Veterans Administration Medical Center, One Veterans Drive, Minneapolis, MN 55417. Phone: 612-725-2098; Fax: 612-727-5640; E-mail: Jaime002{at}tc.umn.edu
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-), a crucial
mediator in sepsis, elicits multiple biologic effects, including intravascular
thrombosis and circulatory shock. TNF- exerts its biologic effects
through two distinct cell surface receptors, TNF-R1 and TNF-R2. The
pathophysiologic interaction between TNF- and nitric oxide (NO) in
glomerular thrombosis caused by endotoxemia in rats and wild-type mice
(C57BL6) as well as in knockout mice that are deficient in TNF-R1 (R1
—/—), TNF-R2 (R2 —/—), or both receptors (R1R2
—/—) was studied. Administration of lipopolysaccharide (LPS;
Escherichia coli endotoxin) resulted in increased NO and TNF-
production but failed to induce glomerular thrombosis. Concomitant
administration of LPS + NG-nitro-L-arginine methyl ester (L-NAME; an NO
synthesis inhibitor) resulted in glomerular thrombosis in rats and in
wild-type mice. Intraperitoneal administration of pentoxifylline before LPS
inhibited TNF- synthesis and prevented glomerular thrombosis in rats
given LPS + L-NAME. In contrast to the results observed in rats and wild-type
mice, administration of LPS + L-NAME did not result in glomerular thrombosis
in knockout mice with either single or double TNF- receptor deletion.
Thus, during endotoxemia, (1) TNF- fosters glomerular
thrombosis if there is deficiency of NO synthesis and (2) both
TNF- receptors are necessary for TNF-'s prothrombogenic action.
Clinically, these novel studies suggest that in gram-negative endotoxemia,
inhibition of NO synthesis and selective blockade of TNF- receptors may
provide unique therapeutic approaches for mitigation of glomerular thrombosis
and restitution of vascular tone. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(INF-), and tumor necrosis factor-
(TNF-)
(1,2);
prostanoids; nitric oxide (NO); and reactive oxygen species such as superoxide
anion and hydrogen peroxide
(3).
During septicemia and as a result of induction of the inducible NO synthase
(iNOS) mediated by LPS (4) and
by cytokines such as TNF-
(5) and INF-
(6), there is production of
large amounts of NO by a variety of tissues, including vascular smooth muscle
(7), glomerular mesangium
(8,9),
and macrophages
(4,10).
NO is a powerful vasodilator that also exerts antithrombotic effects by
inhibiting platelet aggregation and adhesion
(11,12).
Clinical and experimental evidence suggests that the large amount of NO that
is produced during endotoxemia contributes to the development of circulatory
shock (13). In rats with
endotoxemia, inhibition of NO synthesis by substituted L-arginine compounds
such as NG-nitro-L-arginine methyl ester (L-NAME) restores vascular tone but
results in glomerular thrombosis
(14). The latter can be
prevented by concomitant administration of nitroglycerin, an exogenous NO
donor, but not by hydralazine or atrial natriuretic peptide
(15). Hence, this suggests
that during endotoxemia, a certain amount of endogenous NO is necessary to
prevent vascular thrombotic events
(16).
TNF- is one of the crucial inflammatory mediators of endotoxemia and
participates in the genesis of several of its clinical manifestations,
including hypotension, activation of coagulation pathways, and fever
(17,18).
TNF- exerts its actions via activation of two distinct receptors,
namely TNF-R1 (p55) and TNF-R2 (p75)
(19). TNF-R1 mediates most of
the activities classically attributed to TNF-, including circulatory
shock, apoptosis, and fever
(20,21).
The function of TNF-R2 is less well understood, but it has been shown to be
involved in T-cell development and proliferation
(22,23)
and seems to mediate the effects of the membrane-bound cytokine
(24).
TNF- has prothrombotic actions, particularly induction of tissue
factor and thrombomodulin downregulation
(25,26),
that contribute to the development of disseminated intravascular coagulation
during endotoxemia (27).
Disseminated intravascular coagulation is characterized by widespread
deposition of fibrin in the microvasculature, which, in the kidneys, leads to
glomerular thrombosis and renal failure
(1).
Thus, TNF- may play a pivotal role during endotoxemia, given its
prothrombogenic as well as antithrombogenic actions, the latter mediated via
induction of NO synthesis. Given the potential therapeutic implications, we
investigated the pathophysiologic interaction of TNF- and NO in
glomerular thrombosis caused by inhibition of NO synthesis during endotoxemia
(14): (1) in rats, we
studied the effects of TNF- synthesis inhibition with pentoxifylline,
and (2) in knockout mice that are deficient for the type 1, type 2,
or both receptors, we determined the in vivo participation of these
receptors in the genesis of glomerular thrombosis.
|
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were obtained by means of a tail incision 1 h after the LPS administration,
which corresponds to the peak of TNF- production after LPS
administration (28). Three
different groups of rats were studied.
All substances were dissolved in saline and were prepared fresh on the day of the experiments. In preliminary experiments, it was determined that intraperitoneal injections of saline in the same volume and times as described above had no effect on the parameters measured in our study.
At the end of the 8-h experimental period, the rats were anesthetized and killed by exsanguination, and the kidney tissue was prepared for light microscopy as described elsewhere (14). Formalin-fixed, paraffin-embedded 4-µm sections were stained with periodic acid-Schiff (PAS). The PAS sections were examined in a blinded manner, and the percentage of glomeruli that showed any PAS-positive material in the glomerular capillaries was determined (14). At least 50 glomeruli were examined from each kidney.
Studies in Knockout Mice
Wild-type male mice (C57BL6) and knockout mice that lacked the TNF-R1 (R1
—/—), the TNF-R2 (R2 —/—), or both receptors (R1R2
—/—) and weighed between 25 and 30 g were used for all
experiments. Specific pathogen-free wild-type mice (C57BL/6) were obtained
from Harlan Sprague-Dawley. TNF-R knockout mice were obtained from Immunex
Corporation (Seattle, WA) and were bred in microisolator cages at the College
of Veterinary Medicine, University of Minnesota. All breeding pairs were
genotyped by PCR analysis of genomic DNA to confirm TNF-R genotype
(28). All mice were housed in
microisolator cages on a 12-h light/dark cycle and received tap water and
mouse chow ad libitum. The animals were housed in facilities
accredited by the American Association for Accreditation of Laboratory Animal
Care, and the animal studies were approved by the Institutional Animal Care
and Use Committee. Similar to our previous studies in rats and to perform the
studies at the peak of glomerular thrombosis, we performed pilot time-course
experiments. These preliminary experiments suggested that 4.5 h was the
appropriate time to kill the mice. In addition, pilot experiments were
performed to determine the optimal doses of LPS and L-NAME that resulted in
glomerular thrombosis but without increasing lethality. Wild-type and knockout
mice were assigned to two groups.
Separate subgroups of wild-type and knockout mice received an injection of
LPS and were killed 1 h later by retroocular exsanguination. Plasma from each
mouse was saved for TNF- determination.
All agents were dissolved in saline and prepared fresh each day. Four and a half h after being given LPS, mice were anesthetized and killed by exsanguination, and the kidney tissue was prepared for light and immunofluorescence microscopy as described elsewhere (14). Frozen sections were treated with FITC-labeled rabbit anti-human polyclonal antibody against fibrinogen (Dako Corporation, Carpinteria, CA). Slides were examined in a blinded manner, and the percentage of glomeruli that showed positive immunofluorescence or PAS-positive material in the glomerular capillaries was determined. All available glomeruli in each slide were examined.
TNF- Determination
Mouse and rat serum TNF- was by measured by enzyme-linked
immunosorbent assay (R&D, Minneapolis, MN), following the manufacturer's
instructions.
NO2/NO3 Determination
Levels of NO2/NO3, the stable metabolites of NO, were
determined in mouse serum or rat urine by the Griess reaction, as described
elsewhere (9), and expressed as
nmol/ml.
Statistical Analyses
Data are expressed as mean ± SEM. For statistical comparison
involving two groups, an unpaired t test was used, whereas for
comparison involving more than two groups, ANOVA with Sheffe's post
hoc test was used (Statview 512; Abacus Concepts, Inc., Berkeley, CA).
Significance was considered to be present at P < 0.05.
|
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was increased
significantly; pentoxifylline inhibited completely the increase in serum
TNF- (LPS, 80 ± 20% increase over baseline; LPS +
pentoxifylline, 2 ± 3% increase over baseline; P <
0.05)
|
Pentoxifylline Prevents Glomerular Thrombosis in Rats with Endotoxemia and NO Synthesis Inhibition. As was shown elsewhere by us (14), glomeruli from rats that received LPS + L-NAME showed deposition of a PAS-positive homogeneous material within the capillary loops, as well as proteinaceous material within Bowman's space and the tubular lumen, indicative of glomerular thrombosis. No significant cellular infiltration or proliferation was noted within the glomerulus or the interstitium (Figure 2).
|
Conversely, rats that received LPS and had concomitant inhibition of NO
(L-NAME) and TNF- (pentoxifylline) synthesis did not develop glomerular
thrombosis (Figure 2). These
findings therefore demonstrate that a balance between the prothrombogenic
effects of TNF- and the antithrombogenic effects of NO is crucial to
prevent thrombotic events during endotoxemia.
Mouse Experiments
Results from the above experiments, which used the TNF- synthesis
inhibitor pentoxifylline, suggested that TNF- mediates the thrombotic
events of endotoxemia. Pentoxifylline, however, can inhibit endothelial tissue
factor expression, a potent coagulation activator, independent of its effect
on TNF- synthesis (29).
We therefore performed experiments in knockout mice that were deficient for
the TNF- receptors R1 —/—, R2 —/—, or R1R2
—/—.
TNF- and NO2/NO3 Levels. Mice received
an injection of LPS (2 mg/kg), and serum TNF- was measured (n
= 3 to 7 per group). As shown in Figure
3, knockout mice produced similar or higher amounts of TNF-
compared with baseline, therefore suggesting that the absence of TNF-
receptors does not interfere with TNF- production in response to
LPS.
|
Wild-type and knockout mice received an injection of LPS (2 mg/kg), and
serum NO2/NO3 levels were measured at 4.5 h. As shown in
Figure 4, wild-type and
knockout mice produced similar amounts of NO in response to LPS, therefore
suggesting that the lack of TNF- receptors does not prevent the
induction of NO synthesis in response to LPS.
|
Renal Histology. Wild-type and knockout mice that received an
injection of LPS or L-NAME alone showed minimal histologic abnormalities
(Figure 5). Similar to our
studies in rats (14),
glomeruli from wild-type mice that received LPS and L-NAME had increased
deposition of fibrin, as determined by immunofluorescence
(Figure 6) and light microscopy
(Figure 7). However, in
contrast with the results observed in the wild-type mice, neither knockout
mouse (R1 —/—, R2 —/—, or R1R2 —/—)
developed thrombosis after LPS administration and NO synthesis inhibition with
L-NAME (Figures
6,7,8).
These findings therefore suggest that signaling through the two TNF-
receptors (TNF-R1 and TNF-R2) may be necessary for the prothrombogenic effects
of TNF- in endotoxemia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF- (see reference
30 for review). As a result of
iNOS induction, there is production of large amounts of NO for long periods of
time by a variety of tissues, including macrophages, vascular smooth muscle,
and glomerular mesangial cells
(4,8,9).
The large amounts of NO produced, acting in a paracrine and autocrine manner,
participate in the vasoplegia and myocardial depression characteristic of
gram-negative sepsis (13). In
addition to activating guanylate cyclase, NO is oxidized locally to the
inactive and stable metabolites NO2/NO3, which can be
detected and quantified in serum and have been used widely as a surrogate
marker of NO production
(11).
Nonselective inhibition of NO synthesis during endotoxemia restores
vascular tone, but, as shown in our studies elsewhere, this results in
glomerular thrombosis (14),
which can be prevented by the concomitant administration of nitroglycerin, an
exogenous NO donor (15).
Conversely, selective iNOS inhibition does not result in glomerular thrombosis
in rats with endotoxemia (16),
which clearly establishes the importance of endothelial NOS (eNOS) in the
prevention of vascular thrombosis. Given that NO is a powerful inhibitor of
platelet aggregation and adhesion
(12), we surmised that during
sepsis, a certain amount of endogenous vascular NO is necessary to prevent the
thrombogenic cascade unleashed by LPS. TNF- is one of the main
inflammatory mediators in endotoxemia
(30,31).
TNF- is endowed with pleiotropic actions, including the concomitant
induction of iNOS in macrophages
(32), vascular smooth muscle
(33), and glomerular mesangium
(9), as well as promotion of
intravascular coagulation by increasing expression of tissue factor and
downregulation of thrombomodulin
(25). In addition, TNF-
induces the expression of chemokines such as monocyte chemoattractant
protein-1 (34) and adhesion
molecules such as intercellular adhesion molecule-1
(35) and vascular cell
adhesion molecule (36), which
facilitate infiltration and accumulation of macrophages in the glomerulus
(37) and promote thrombosis
(38). However, studies in
vivo (39) and in
vitro (33) have
demonstrated that TNF- downregulates eNOS expression. TNF-
downregulates eNOS expression by increasing the rate of eNOS mRNA degradation
through a process that requires new protein synthesis
(40). At the same time,
TNF- upregulates NO production from iNOS in other tissues, including
vascular smooth muscle and the glomerulus
(4), resulting in the
characteristic vasoplegia of gram-negative sepsis
(13).
A number of clinical trials have attempted to abrogate septic shock by
inhibiting TNF-, by use of either antibodies
(41) or fusion proteins
composed of the extracellular domain of TNF-R1 or TNF-R2
(42,43).
However, data are conflicting and clinical responses are disappointing.
Possible explanations include differences in trial design and incomplete
knowledge of the cellular events triggered by TNF- and the TNF-
receptors involved (44).
In our studies, we first demonstrated that treatment with pentoxifylline
inhibited TNF- synthesis in response to LPS. Inhibition of TNF-
in rats with endotoxemia and NO synthesis inhibition prevented glomerular
thrombosis in these animals, which suggests that a balance between the
prothrombogenic effects of TNF- and the antithrombotic effects of NO is
crucial to prevent the thrombotic events of endotoxemia. The effects of
TNF- are mediated via activation of two TNF- receptors, TNF-R1
and TNF-R2 (19). The
availability of knockout mice that lack either receptor or both allowed us to
explore in the current studies the role of these receptors in both the
induction of NO synthesis and the prothrombogenic actions of TNF- in an
in vivo model of endotoxemia. We first demonstrated that, similar to
our studies in rats (14),
L-NAME inhibition of NO synthesis in wild-type mice results in glomerular
thrombosis only in mice previously exposed to LPS. Conversely, knockout mice
that were deficient in either one or two receptors failed to develop
glomerular thrombosis after introduction of LPS + L-NAME, despite that
knockout mice manifested similar increases in serum TNF- to those shown
by wild-type mice. The serum levels of NO2/NO3, stable
metabolites of NO, increased similarly in wild-type and knockout mice. This is
not unexpected, because LPS stimulates the production of other cytokines such
as IL-1, IL-2, IL-6, and INF- that, in addition to TNF-
(45), contribute to the
induction of iNOS (13).
Furthermore, studies in knockout mice that lack the TNF- receptors have
shown normal upregulation of INF-, TNF-, and IL-1 and preserved
iNOS induction during murine toxoplasmosis as well as in response to LPS
(46,47,48).
These findings are important because, in response to LPS, TNF- is
synthesized and released locally in glomeruli, and most of its actions are
mediated in a paracrine manner
(49). Indeed, previous studies
have demonstrated clearly the local synthesis of both NO
(4) and TNF- in
glomeruli of animals exposed to LPS
(50).
Recent studies have shown that selective iNOS inhibition with preservation
of eNOS activity prevents the fall in BP, reduction in GFR, and glomerular
thrombosis in rats with endotoxemia
(16). These findings suggest
that NO produced by eNOS is critical to prevent the hemodynamic and thrombotic
events of endotoxemia. Currently available iNOS inhibitors, however, are not
completely selective and therefore are not suitable for use in clinical
trials. Nonetheless, on the basis of the current studies, it is tempting to
speculate that in gram-negative sepsis, restoration of vascular tone with NO
synthesis inhibitors may be achieved without promotion of glomerular
thrombosis, if there is concomitant blockade of the TNF- receptors.
|
Acknowledgments |
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measurements, and to Barb Devereaux and Betty Mart for secretarial
support. |
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
Am J Physiol 261:C634
-C641, 1991 on
nitric oxide synthase activity and endothelin production by vascular
endothelial cells. J Clin Invest90
: 879-887,1992
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