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Animal Model |
From the Cardiovascular Biology Program,*
Oklahoma
Medical Research Foundation, and the Department of
Pathology,
University of Oklahoma Health
Science Center, Oklahoma City, Oklahoma; the Department of Medical
Microbiology and Immunology,
Texas A&M
University Health Science Center, College Station, Texas; and the
Department of Pathology,§
and Division of
Nephrology,¶
Department of Pediatrics,
University of Utah School of Medicine, Salt Lake City, Utah
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5
ng/ml) in the general circulation. Light and electron microscopy showed
organelle disintegration and necrosis of the renal proximal tubular
epithelium and of the intestinal mucosal epithelium at the tips of the
microvilli, both of which were previously shown to bear
Gb3 receptors. The renal distal tubular epithelium was
spared. The renal proximal tubular epithelial changes were accompanied
by swelling of visceral epithelial cells (podocytes) and by swelling
and detachment of endothelial cells of the glomerular capillaries. In
addition, all of the animals receiving low-dose Stx-1 showed
microvascular fibrin deposition and thrombosis in renal glomerular and
peritubular capillaries in association with a fall in hematocrit and
platelet count and a rise in schistocyte count. The gastrointestinal
villous tip lesions were accompanied by varying degrees of mucosal and
submucosal congestion, hemorrhage, or necrosis.
Electron microscopic images of cerebral cortex and cerebellum showed
diffuse unraveling of myelin sheaths with occasional disintegration of
neuronal cell bodies. In contrast to the gastrointestinal mucosal and
renal proximal tubular epithelium, the Gb3 receptor
glycolipid of the renal glomerular and neuronal tissues as determined
using toxin overlay thin-layer chromatography plates was below the
limit of detection (<13 pM/g wet tissue). We conclude that,
depending on the status of the host and amount of toxin
infused, Stx-1 can produce a variety of
responses ranging from damage to cells carrying the
Gb3 receptor (renal proximal tubular epithelial cells and
gastrointestinal mucosa) to damage to renal glomerular tissues with
microvascular thrombosis as a result of the host's inflammatory
response localized to the kidney. We conclude that this thrombotic
coagulopathy arises from local changes in the kidney because the
appearance of host inflammatory mediators was limited to the urine.
This suggests that the initial host response is localized in the
kidney, and that the systemic thrombocytopenia,
anemia, and schistocytosis may arise
secondarily.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite impressive advances in the biochemistry and genetics of SLTs, the precise roles of SLTs and host immune response to the toxins in the development of bloody diarrhea and extra-intestinal sequelae remain to be elucidated. Histopathological examination of tissues from patients with SLT-associated acute renal failure (HUS) or thrombotic thrombocytopenic purpura (TTP) usually show striking microvascular lesions. In the kidney, glomerular endothelial cells are swollen and detached from the glomerular basement membrane, and glomerular capillaries frequently contain fibrin thrombi.6-8 Thrombocytopenia and the presence of fragmented erythrocytes (schistocytes) on blood smears also are observed. The constellation of acute-onset oliguria or anuria, azotemia, microangiopathic hemolytic anemia, and reduced platelet counts following prodromal diarrheal illness define the diarrhea-associated hemolytic uremic syndrome (D+ HUS).
Based on these clinical studies, the concept emerged that SLTs
specifically target microvascular endothelial cells for damage.
However, human endothelial cells derived from umbilical or saphenous
veins are not sensitive to low concentrations of purified SLTs unless
cultured in the presence of SLTs plus the pro-inflammatory cytokines
tumor necrosis factor-
(TNF-) or interleukin-1
(IL-1).9,10
Cytokine-mediated sensitization of endothelial
cells is associated with increased synthesis and membrane expression of
the toxin receptor Gb3.11,12
Monocytes may
participate in pathogenesis, as human monocytes also synthesize TNF-
and IL-1 when incubated with purified SLTs in
vitro.13
Although these data suggest that SLTs and/or
endotoxin-induced host factors target microvascular endothelial cells
for damage, immunofluorescence staining of human kidney tissue for SLT
binding suggests that Gb3 is primarily localized to
proximal tubules rather than glomeruli.14,15
Although
histopathological studies of patients and in vitro
experiments have been useful in developing hypotheses regarding the
pathogenic role(s) of SLTs in hemorrhagic colitis and D+ HUS,
experimental evaluation of these hypotheses is hampered by the lack of
experience with a subhuman primate model. Studies carried out using
mice, rabbits, and pigs have supported the concept that the sites of
elevated toxin receptor expression coincide with sites of tissue
damage. Each of these animal models, however, has certain
limitations.16
Experience with a subhuman primate model may
provide information under controlled conditions concerning the range
and variability of responses to Stx-1 that would be directly applicable
to the disease as seen in humans. Using immunofluorescence and
thin-layer chromatography techniques, we previously demonstrated that
the sites and quantities of Gb3 in human and baboon
(Papio anubis cynocephalis) kidney tissues are
similar.15
We report here additional experiments
characterizing the baboon as an animal model for the study of Stx-1
(SLT-1) in which the following questions are addressed. 1) What are the
responses to high and low concentrations of Stx-1 in the baboon, and do
these responses mimic the hemolytic uremic syndrome (ie, acute onset of
oliguria, azotemia, microangiopathic hemolytic anemia, and
thrombocytopenia with histopathological evidence of glomerular
capillary endothelial swelling and fibrin/platelet deposition)? 2) Are
the proximal tubular and gastrointestinal mucosal epithelium, which
contain the Gb3 receptors, affected directly by the toxin?
3) Are adjacent structures (eg, glomerular capillary endothelium and
podocytes) affected? 4) To what degree are either of these categories
of responses (direct toxin versus host mediated) limited to
the kidney and gastrointestinal tract, and to what extent are other
systems, including the hemostatic, inflammatory, and central nervous
systems, affected in this model?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified Stx-1 was prepared from cell lysates of E. coli transformed with plasmids containing the structural genes under control of the phage T7 promoter.10,17 Stx-1 was purified by sequential chromatography over anion-exchange, chromato-focusing, and immunoaffinity columns. To reduce endotoxin contamination, Stx-1 preparations were passed over Cibachrome blue columns. All toxin preparations contained <0.1 ng of endotoxin per ml as assessed by Limulus amoebocyte lysate assay. The specific cytotoxicity of the toxin preparations was 2 x 106 CD50/µg of protein as assessed by vero cell cytotoxicity and Pierce protein quantitative kits. Toxin preparations were stored at 4°C. Dilutions of Stx-1 were prepared in sterile saline immediately before each experiment.
Pre-Experimentation and Experimentation Procedures
Papio c. cynocephalus or Papio c. anubis baboons were purchased from a breeding colony maintained at the University of Oklahoma Health Sciences Center or from Biomedical Research Foundation, Houston, TX. Animals weighed 3.4 to 15.5 kg, had leukocyte concentrations of 5000/mm3 to 14,000/mm3, and hematocrits exceeding 36%. They were screened for tuberculosis. These animals were held for 30 days at the University of Oklahoma Health Sciences Center animal facility, and the infusion studies were done in this facility. The animals were observed continuously during the first 2 hours after infusion. All animals were followed until they died or were moribund, at which time they were euthanized. The kidney, small and large intestine, heart, lung, liver, spleen, adrenal, pancreas, and brain were examined, and specimens were collected for light microscopy within 1 hour of death. Samples from kidney, small and large intestine, and brain also were collected for electron microscopy. This study protocol received prior approval by the Institutional Animal Care and Use Committee of both the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center.
Infusion Procedures
The baboons were fasted overnight before the study, allowed water ad libitum, and immobilized the morning of the experiment with Ketamine (14 ± 0.5 mg/kg intramuscularly). Sodium pentobarbital (2 mg/kg) then was infused every 20 to 40 minutes. The animals were intubated orally and allowed to breathe spontaneously.
A superficial femoral vein was exposed and cannulated aseptically and
the catheter advanced to the inferior vena cava to sample blood and
monitor central venous pressure. The femoral artery was cannulated to
monitor arterial pressure. Each baboon was placed on its side in
contact with a controlled temperature heating pad. Blood pressure and
rectal temperature were monitored using a Statham pressure transducer
and Gould recorder (Valley View, OH), and a telethermometer (Yellow
Springs Instrument Co., Yellow Springs, OH), respectively. A
percutaneous catheter in the cephalic vein was used to infuse saline or
purified Stx-1 and sodium pentabarbital. Either saline, a high
dose (2.0 µg/kg), or a low dose (0.05 to 0.2 µg/kg) of Stx-1 was
infused as a bolus into 18 animals (see Table 1
).
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Sampling and Monitoring Procedures
Blood and urine samples were obtained, and monitoring of vital signs and urine output was recorded at T-0 and at 2, 6, 12, 24, 36, 44 to 48, and 52 to 56 hours. The whole blood samples at each drawing included 1.0 ml anticoagulated with EDTA for complete blood count (CBC); 2.0 ml anticoagulated with 3.8% sodium citrate for fibrinogen,18 prothrombin time, tissue plasminogen activator (t-PA),19 thrombin-antithrombin complex (TAT),20 factor VIIa,21 TNF, and IL-622; 1.0 ml in trasylol/thrombin for fibrin degradation products (FDP)23; and 1.0 ml of clotted blood for blood urea nitrogen,24 creatinine,25 serum glutamic pyruvic transaminase,26 and electrolytes. Not more than 10% of the animal's blood volume was collected during the first 12 hours. Urine was collected over a 1-hour period at each of the time intervals listed above via a Foley catheter, and the volume of urine produced during this hour was measured together with urine protein, hemoglobin, pH, glucose, and acetone. Aliquots of urine (1 to 2 ml) were frozen at -80°C for assay of TNF, IL-6, and FDP. Urine TNF and IL-6 were determined using commercial (Pharmingen, San Diego, CA) ELISA kits.
Precautions against development of hypovolemia were taken by measuring central venous pressure at each of these intervals and replacement of fluids (saline) using the following criteria: 1) weight less than control (initial period) weight: infuse sufficient normal saline to return animal to initial weight (1 g = 1 ml) at 1 ml/kg/minute; 2) blood pressure (BP) low (mean pressure <75): infuse normal saline, 10 ml/kg (1 ml/kg/minute), repeat as needed while following CVP and BP, and stop when mean BP >75, or sooner if CVP rises above 10); 3) CVP <3: infuse normal saline at 10 ml/kg (1 ml/kg/minute), and repeat as needed to raise CVP >3; 4) urine output <2 ml/kg/hour (0.5 ml/kg/15 minute) but weight, BP, and CVP are not low: infuse 10 ml/kg normal saline (1 ml/kg/minute) only once, and do not give if CVP >10.
Light Microscopy (LM) and Transmission Electron Microscopy (TEM)
Fixation
Representative tissue samples of each organ listed above were fixed in 10% neutral buffered formalin for routine histopathology and in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for plastic embedded light and electron microscopy. Sampling protocol for renal tissue included collection of tissue (coronal section) from both kidneys at three different sites (lower, mid, and upper poles). See below for the predetermined scoring system of the light microscopic observations. Sampling protocol for gastrointestinal (GI) tract tissues involved sampling of those areas that were visibly involved.
Processing for LM
For LM, tissues were fixed in neutral buffered formalin for at least 24 hours, processed by standard methods, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and phosphostungstic acid hematoxylin (PTAH).27,28 To illustrate the differential responses among animals and among organs within the same animal, tissues from animals exposed to a high concentration of toxin (2.0 µg/kg) were compared with those that received low concentrations of toxin (0.05 to 0.2 µg/kg). Gut microvilli were examined for histopathological changes in the villous tip, lacteal, villous base, and crypt. Renal cortex was examined for histopathological changes in the glomerular and proximal, distal, and collecting tubules. The outer renal medulla and papilla also were examined. The brain was examined for histopathological changes involving the cerebral cortex and pons.
Processing for TEM
Tissue samples (1 mm thick) fixed in 2.5% glutaraldehyde in PBS, pH 7.4, for 72 hours in the refrigerator were post-fixed in 2% OsO4 in water for 1 hour, washed in PBS followed by distilled water, and then en bloc stained for 60 minutes in 2% uranyl acetate and 0.25% lead citrate in distilled water. The samples were dehydrated in a graded ethanol series, infiltrated overnight with a 50:50 mixture of propylene oxide and Embed 812 (Electron Microscopy Science, Ft. Washington, NY), and then infiltrated with pure resin for 6 to 8 hours.29-32 Finally, the specimens were transferred to Beem capsules filled with pure resin and polymerized in a 60°C oven for 16 to 24 hours. Thick (1-µm) sections from each block, stained with Paragon multi-stain (Paragon C&C Co., Bronx, NY) and mounted in Permount (Fisher Scientific, Pittsburgh, PA) were used to prepare survey photographs and to select areas for EM thin sectioning. Thin sections (silver to gray) stained for 4 minutes in 5% uranyl acetate in 25% methanol and 4 minutes in 0.3% lead citrate in water, both at room temperature, were examined in a Hitachi MET scope or a JOEL JEM 1200 EX-II.
Statistical Analysis and Scoring of Histopathology
Statistical Analysis
The clinical data were analyzed using an analysis of variance with Duncan's multicomparison test to determine significant differences (P < 0.05) between groups at given times and for certain variables. An analysis of variance, repeated measure design, with Dunnett's multicomparison test was also used to determine significant differences (P < 0.05) between time 0 (T-0) or baseline and subsequent times for a given group.
General Evaluation of Histopathology of All Organs
The pathological lesions of adrenal glands, kidneys, and lungs were analyzed by dividing their description into five categories: congestion, white cell influx, hemorrhage, thrombosis, and necrosis. The tissues were rated according to the severity of the histopathological lesions. The scale ranged from 1 to 4, with 4 being the most severe. All microscopic sections were read by Dr. Kosanke, who was blinded as to which study was being analyzed. The Kruskal-Wallis test, a nonparametric test, was used to determine significant differences (P < 0.05) between groups for a given pathological lesion.
Specific Evaluation of Renal Histopathology
One slide from each case was examined at x400 magnification until 100 consecutive glomeruli were assigned to one of eight categories: no thrombotic microangiopathy (TMA), endothelial swelling (ES), fragmented red blood corpuscles (FR), fibrin thrombi (FT), endothelial swelling with fragmented red blood corpuscles (ES + FR), endothelial swelling with fibrin thrombi (ES + FT), all three lesions (ES, FR, FT). All microscopic sections were read by Dr. Pysher who was blinded as to which study was being analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical, Routine Laboratory, and Histopathological Findings
We studied three different groups. These included a control and
two groups receiving high and low concentrations of Shiga-like toxin 1
(Stx-1) (Table 1)
.
Control Group
The control group showed no significant changes in vital signs or
markers of renal function (urine volume, protein and blood, creatinine,
and potassium) or hemostatic activity (platelet count and fibrin
degradation products). The total white cell count did rise from
8.7 ± 1.5 x 103
cells per mm3
at
T-0 to a peak 17.6 ± 0.1 at T + 6 hours. The count returned
to baseline by T + 24 hours. No schistocytes above 1% were
observed, and there were no significant changes in hematocrit. Table 2
shows evidence of only a slight degree
of congestion in the tissues examined and, in the case of the lung, a
modest influx of white cells. Table 3
shows that an average of 86.6 ± 3.9% of glomeruli had no
evidence of thrombo-microangiopathy (TMA) with only 12.8 ± 3.7%
showing any evidence of endothelial swelling. All animals of this
control group were sacrificed at T + 72 hours.
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Table 4
shows that the average
response of animals 1, 2, 13, 14, and 15 to high-dose Stx-1 (2.0
µg/kg) was characterized by 4+ proteinuria and by a sharp fall in
urine volume from 2.3 to 0.7 ml/kg/hour by T + 12 hours followed
by anuria. This was accompanied by creatinine and anion gap (GAP)
peaking at 5.9 mg/dl and 36 mmol/L, respectively, and by severe
hyperkalemia peaking at 15 mmol/L by T + 44 hours. The blood
hematocrit, however, was stable at 41% from T-0 to T + 44 hours.
There was no increase in schistocytes. In the kidney, the light
histopathological changes were less pronounced in this group challenged
with high concentrations of toxin than in that receiving low
concentrations of toxin. Table 2
shows evidence of significant
congestion and white cell influx in the adrenals, lungs, and liver. In
addition, tissue from the GI tract (both small and large bowel)
exhibited severe mucosal and submucosal hemorrhage, thrombosis, and
necrosis. Table 3
shows that an average of 61.8 ± 4.5% of
glomeruli had no evidence of TMA with only 28.7% and 6.3% exhibiting
endothelial swelling and fibrin thrombi, respectively. The gross
examination of the brains of the high-dose animals at the autopsy
showed edema as reflected by flattened sulci. Light microscopy showed
little or no evidence of injury to neuronal tissue of either gray or
white matter in the cerebral cortex, pons, or the floor of the fourth
ventricle. Survival time of the high-dose group averaged 35.4 ±
4.3 hours.
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Table 5
shows that the response of
animals 3 to 6, 9, and B9 to B11 to low-dose Stx-1 (0.05 to 0.2
µg/kg) also was characterized by a sharp fall in urine volume from
4.0 and 6.0 ml to 0.7 ml by T + 24 hours. This was accompanied by
proteinuria at T + 24 hours and by the creatinine concentration
and anion gap peaking at 2.0 mg/dl and 27.7 mmol/L, respectively, at
T + 52 hours. The potassium concentration peaked at 5.2 mmol/L at
T + 52 hours. This was accompanied by an increase in schistocytes
from 2.7% to 8.5% during this interval. In the kidney, the
histopathology was more pronounced in animals challenged with low
concentrations of toxin than those receiving high concentrations of
toxin. Table 2
shows evidence of significant congestion and white cell
influx in all organs studied. In addition, hemorrhage was observed in
tissues from the GI tract and adrenal glands. The GI tract lesions were
much less severe than those observed in the high-dose group. Of
particular significance in this group was the evidence of thrombosis
and necrosis in the renal tissues. Table 3
emphasizes the importance of
these renal findings by showing that on a systematic examination of
these tissues for histological markers of renal involvement, only
7.3 ± 2.2% of glomeruli had no evidence of TMA with 39.9% and
41.3% exhibiting endothelial swelling and combined endothelial
swelling and red corpuscle fragmentation, respectively. The brains of
low-dose animals had normal gross appearances and weight. They were not
submitted to microscopic or ultrastructural examination. Survival time
of the low-dose group averaged 57 ± 3.5 hours.
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).
Fibrinogen concentration of both groups rose and peaked at 44 to 48
hours at concentrations ranging from 135% to 1987% of baseline
whereas fibrin split products ranged from a peak of 22 µg/dl in the
high-dose group to 78 µg/dl in the low-dose group. These changes
involving hemostatic components were accompanied by a fall in platelet
count to 45 x 103/mm3
or below in both
the high- and low-dose groups. The adrenals of animal 5 of the low-dose group were the most severely affected of all the animals in this study. This severity of the adrenal lesion was unique to this one animal. The lungs and liver of all animals showed moderate congestion and mild to moderate leukocyte infiltration.
Light and Electron Microscopic Pathology of Renal, GI, and Central Nervous Systems
Renal Glomeruli
Figure 1
compares the
glomeruli of control animals (Figure 1A)
with those from the high- and
low-dose Stx-1 groups. The glomeruli from the high-dose group (Figure 1B)
are congested but exhibit no thrombotic angiopathy, whereas the
glomeruli from the low-dose group (Figure 1C)
show almost universal
obliteration of the capillary lumina, frequent fragmentation of red
blood corpuscles, and occasional fibrin thrombi. The tubules of control
animals (Figure 1D)
show no epithelial necrosis, whereas those of the
high-dose group (Figure 1E)
show extensive necrosis, and those of the
low dose group (Figure 1F)
show an intermediate degree of epithelial
necrosis. Figures 2A and 3a
(1-µm plastic sections) show
glomeruli from the high-dose group (animal 13) congested with blood in
the capillary loops, some endothelial cell swelling, and generalized
edema with reduction of the urinary space within the glomerular
capsule.
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, b and c (1-µm plastic sections), show
glomeruli from the low-dose group (animal 9) with more extensive
lesions than the high-dose animals. Fibrin clots were a common finding
in the capillary loops; however, there was no swelling of the tuft and
there was retention of ample urinary space within Bowman's capsule. By
electron microscopy, the glomeruli from this low-dose group showed
nonocclusive fibrin deposits in the capillary loops and osmophilic
inclusions in the visceral epithelial cells. All members of this
low-dose groupexhibited positive immunostaining for fibrin deposits in
renal glomeruli and fragmented red blood corpuscles (Figure 4, A and B)
.
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The most severely affected segment of the nephron was the proximal
tubule (Figure 5)
. The figures illustrate
the representative changes that occur in the high-dose animals. The
minimal changes seen were increased clear vacuoles in the tubular
epithelium (Figure 5a)
but with normal-appearing brush borders and
peritubular capillaries. Early signs of tubular epithelial degeneration
included shortening or loss of the brush border and occa-sional
blebbing of epithelial cytoplasm into the tubule lumen (Figure 5c)
. One
of the more common features of the proximal tubules from animals
exposed to high concentrations of toxin was the presence of casts in
the lumen of otherwise normal or near-normal proximal tubules (Figure 5e)
.
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. Frank extrusion of epithelial cytoplasmic
blebs was a frequent finding (Figure 5d)
. These cytoplasmic blebs
varied in their size and content (some with cytoplasmic organelles and
some without). The principal feature of the tubules in this low-dose
group was the widespread damage (Figure 5f)
in tubular epithelium that
made it difficult to identify the tubule type. This type of damage was
observed in all three zones examined (ie, cortex, outer medulla, and
papilla). The distal tubules in all three groups were spared. GI Tract
In contrast to the renal tubules, the degree of damage to
intestinal mucosal villi was directly dose dependent (ie, the high-dose
animals showed the highest degree of villous damage of both small and
large bowel; Figure 6, a and c
), and the
low-dose toxin group showed significantly less damage (Figure 6, b and d)
. The most common features of the damaged villi in the high-dose
toxin group were the loss of epithelial cells from the tip of the villi
(Figure 6a)
and blood-filled lacteal (Figure 6c)
. By comparison, the
villi of the low-dose toxin group had normal and intact epithelium even
at their tips (Figure 6b)
and only minimal changes in the submucosa,
ie, mild increase in macrophages near the tip (Figure 6d)
.
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Electron microscopic studies revealed subtle but significant
changes in the brains of high-dose animals, including the separation
(unraveling) of myelin of the larger nerve fibers in all areas examined
(Figure 7, A and B)
plus perivascular
edema (Figure 7B)
. The microvessels were normal appearing in all
respects. The mitochondria and glial fibrils appeared normal,
indicating adequate fixation and preservation during the EM processing.
In some areas of the brain large neuronal cells and some glial cells
showed degeneration. However, the degeneration was focal and spotty,
ie, degenerating neuronal cell body and two adjacent glial cells, one
degenerating and one intact. This latter phenomenon was also observed
in absence of degeneration of the adjacent neuronal cell body. Here,
too, it is important to note that the mitochondria were normal in all
neuronal cells but the degenerating ones.
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Table 6
shows that concentrations of
TNF and IL-6 in the urine increased from 1055 to 4609 pg/ng and 434 to
4463 pg/ng creatinine, respectively, whereas the blood value of IL-6
increased only slightly during the same interval. The T-0
concentrations of TNF and IL-6 in the urine of normal baboons are <25
and 15 pg/ng creatinine, respectively.
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The inflammatory response of the two experimental groups as
reflected by the white cell count was remarkably mild (see Tables 4 and 5
). Only the high-dose group showed an elevation in white cell count
with a shift to the left at T + 6 hours. The lack of appearance of
cytokines in the plasma in any of these groups also suggests a limited
systemic inflammatory response. The appearance, however, of an
acute-phase protein response as reflected by a two- to fivefold rise in
fibrinogen in both groups probably reflects a low-grade inflammatory
response of increasing intensity over time.
In contrast, the response of the hemostatic components was more revealing. Although no TAT complexes appeared in plasma, there was a reduction in factor VIIa concentrations in the low-dose but not in the high-dose group. The factor VIIa concentration fell as low as 53% of baseline before returning to normal. The fibrinogen concentrations, however, of both groups rose and peaked at 44 to 48 hours at concentrations ranging from 135% to 1987% of baseline while fibrin degradation products ranged from a peak of 22 µg/dl in the high-dose group to 78 µg/dl in the low-dose group. The rise in plasma t-PA concentrations, however, was the highest in the high-dose group. This is in contrast to the serum fibrin degradation product concentrations, which were the highest in the low-dose group. The response of hemostatic factors of neither the high- nor the low-dose group met criteria for disseminated intravascular coagulation (DIC) as the fibrinogen concentration rose and prothrombin times (not shown) remained stable in both groups (the prothrombin times of the high- and low-dose groups ranged from 12.1 to 13.2 and 12.6 to 14.1, respectively, within our normal range of 11.8 to 13.8 seconds). These relatively limited changes in the hemostatic factors, however, were accompanied by a fall in platelet count to 45 x 103/mm3 or below in both groups by 44 to 48 hours.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The association of proximal tubular epithelial and the GI mucosal/submucosal injury in all groups corresponded with the presence of Gb3 receptors for Stx-1 and is in agreement with observations of numerous investigators using other animal species.34-40 In particular, our observation that bolus intravenous infusion of Stx-1 damages villous capillaries with the appearance of red cells in the extravascular space is in accordance with the earlier studies of Fontaine et al.40 They showed that macaque monkeys fed intragastrically with a nontoxigenic strain of Shigella dysenteriae developed dysentery without the destruction of capillary loops within the colonic mucosa characteristic of ingestion of wild-type Shigella dysenteriae. Our observations of a 12-hour lag period together with an inflammatory response that was limited to the rise in fibrinogen concentration suggests that despite Stx-1 being cleared rapidly37 and having relatively high affinity (4.6 x 10-8 mol/L) for Gb3,41 its direct effects on target tissue develop slowly. This may reflect the fact that the toxin through its glycosidase activity may require time to inhibit protein synthesis.42-45 Recent observations also have shown that verotoxins can induce an increase in prepro-ET-1 mRNA at a concentration of toxin that causes little or no effect on protein synthesis.46
The renal glomerular lesions and the gut submucosal hemorrhagic lesions are not as readily explained as are those involving the renal tubular and intestinal epithelial cells that carry the Gb3 receptors for Stx-1. Studies have shown, however, that human endothelial cells will respond to Stx-1 if they are co-incubated with TNF or IL-19,10 and that these cells under these conditions will synthesize and express Gb3.11 Other investigators47,48 have observed that human renal tubular epithelial cells are capable of producing cytokines in response to endotoxin (LPS), which raises the possibility that cells adjacent to the renal glomerular endothelium could in some as yet described manner supply the cytokines that are thought to be necessary to induce expression of Gb3 by endothelial cells. Studies by Karpman et al suggest that endotoxin and/or host cytokines work in conjunction with Stx-1 produced by E. coli 0157-H7.49 They fed LPS-responder mice (C3H/HeN) and LPS-nonresponder (C3H/HeJ) mice E. coli 0157-H7 intragastrically and observed that the responder mice developed a severe combination of GI, neurological, and systemic symptoms, whereas the nonresponder strains developed a biphasic course of disease with milder symptoms followed later by neurological symptoms. They concluded that although both strains exhibited symptoms to the toxin, the difference in response between the two strains could be ascribed to the combined effects of toxin and LPS in the responder mice and to toxin alone in the nonresponder strain.49 Our studies demonstrate that cytokines (TNF and IL-6) appear in the urine beginning at 12 hours after Stx-1 infusion and that at no time do they appear in quantities above 5 ng/ml in the plasma. This suggests, but does not prove, that these cytokines are produced locally and that there is a local but unequivocal host inflammatory response to Stx-1. This coincides with the above referenced studies suggesting that toxin and host inflammatory mediators could, acting together, expand the specific targeted effect of the toxin to involve adjacent tissues such as the glomerular visceral epithelium and capillary endothelium (in addition to proximal tubular epithelium) in the case of renal lesions, and the GI submucosa (in addition to the epithelium of the mucosal villous tips) in the case of the GI tract lesions. In neither case have these postulated events been established in vivo.
The observation that the low-dose group exhibited the hemolytic uremic syndrome suggests that the above described events can be expanded to produce a dysfunctional glomerular capillary endothelium with perturbation of hematological and hemostatic elements. In addition to the light and electron microscopic pathology described above, this group of animals exhibited irreversible thrombotic lesions of the glomerular capillaries and the most clearly defined systemic signs of perturbation of the hemostatic system. Although much of the glomerular capillary endothelium appeared intact there were foci where the endothelium was edematous, formed blebs, and was either retracted at the intercellular junctions or completely separated from the subendothelium. Fibrin thrombi were observed both on intact endothelium and on exposed basement membrane as reported by others.8 There were some instances where platelets were observed outside these thrombi. Changes in the circulating hemostatic factors in the low-dose group did not begin until 24 hours at which time the platelet count and hematocrit fell. This was followed at 36 hours by the appearance of schistocytes and by the fall in concentration of factor VIIa. This suggests that the components of the hemostatic system did not participate in the chain of events leading to renal failure and uremia in the low-dose group. There was, however, evidence of perturbation of endothelium as reflected by the rise in tissue plasminogen activator concentration beginning at 24 hours. This was accompanied by up-regulation of the production of fibrinogen (acute-phase protein) and the appearance of fibrin degradation products, both of which peaked at T + 42 to 52 hours in the low-dose group. These peak fibrinogen concentrations tended toward being higher in the low-dose than in the high-dose group, although they did not meet statistical criteria for significance. In short, the profile of hemostatic activity in this low-dose group reflects a microangiopathic thrombosis with thrombocytopenia, a decline in hematocrit coupled with the appearance of schistocytes, and evidence of fibrinolytic activity (t-PA and FDP) in the absence of any systemic evidence of fibrinogen consumption or the appearance of thrombin-antithrombin complexes. Although there is evidence of systemic hemostatic activity, this appears to be a localized thrombotic coagulopathy as opposed to a systemic consumptive coagulopathy. This low-dose group accurately reflects what is usually seen in childhood post-diarrheal hemolytic uremic syndrome.50 The lack, however, of a GI prodrome in this intravenous model of HUS may exclude the potentially important parallel pathophysiological effects of endotoxin originating from the gut. Finally, the observation that factor VIIa concentration fell and reached its nadir at T + 24 to 48 hours is a new observation. It suggests that the tissue factor/VII complex can, together with platelets, contribute to the microangiopathic thrombotic response whether it is expressed by an intact but dysfunctional endothelium or exposed by an endothelium that has retracted or desquamated. This has been observed after infusion of endotoxin into humans51 and has led to the speculation that tissue factor exposed or expressed as a consequence of reperfusion might act as a sink for factor VIIa during the 24- to 48-hour interval. This decline in factor VIIa concentration has not been observed, however, in patients with hemolytic uremic syndrome.52
A surprising observation was the electron microscopic evidence of lesions in the central nervous system in the absence of focal neurological signs or brain Gb3 receptors. Although gross examination revealed evidence of cerebral edema in the high-dose toxin group, the light microscopic studies revealed no structural abnormalities. In contrast, electron microscopic studies revealed an unraveling or separation of myelin sheath filaments uniformly throughout the central nervous system and focal areas of neuronal body degeneration. These findings were consistent with those of Fujii et al53 in the mouse and Mizuguchi et al54 in the rabbit. Interestingly, when Zoja et al38 performed toxin overlay assays on chromatographically separated rabbit brain neutral glycolipids, they did not detect toxin binding to Gb3; rather, only faint Stx-1 binding was detected in the ceramide dihexoside fraction. In accordance with this earlier study, our analysis of baboon neuronal tissue for Gb3 receptor detected as little as 10 ng or 13 pmol/L Gb3/mg wet weight of neuronal tissue using toxin overlay thin-layer chromatography plates. The affinity of Stx-1 for Gb3 is estimated as 4.6 x 10-8 mol/L. Thus, assuming Stx-1 pentamer could pass the blood-brain barrier, it is possible that a productive interaction with picomolar quantities of Gb3 or other neutral glycolipid species could produce the lesions observed. Alternatively, as with the renal pathology, host inflammatory mediators released locally could contribute to neuronal tissue injury. This possibility is supported by the observation that low but detectable concentrations of IL-6 were found in the spinal fluid of rabbits and the fact that astrocytes as well as microglial elements can produce cytokines.55-59 Cytokines themselves can cause inflammatory pathology in the brain.59-63 They are cytotoxic to oligodendrocytes in vitro 64-66 and can alter the permeability of the blood-brain barrier capillary endothelium.67 The actual steps involved in the production of these lesions are not clear from these studies. The absence of leukocyte infiltration has been described in the early stages of experimental meningitis,68-70 and the absence of elevated blood levels of cytokines, however, does favor injury due to toxin/cytokine interactions localized to the central nervous system.
The following conclusions are emphasized. First, the injury to renal tubular and glomerular structures can occur independently of microangiopathic thrombosis. The injury to these structures appears to arise from direct toxin damage as well as inflammatory mediator activity. Of special interest is the observation that the production of these mediators appears to be localized to the renal tissue. Second, although microangiopathic thrombosis may not be a necessary sole source of renal and GI tract pathology, the response, when it occurs, can amplify the initial injury (irreversibly). This response not only is driven by platelets but may also be driven by the expression of tissue factor by a dysfunctional capillary endothelium as reflected by the consumption of factor VIIa in these studies. Third, involvement of the central nervous system recalls anecdotal observations made in clinical practice50 and the observations of Fujii et al53 and Mizaguchi et al.54 Again, like the renal phenomenon, the central nervous system response may also be localized in response to host mediators.
Finally, we believe that this primate model in which a low-dose of Shiga-like toxin is infused as a single bolus mimics the hemolytic uremic syndrome, absent the initial diarrheal prodrome. We believe that it offers a base for future studies of mechanism and of conditions (route, method, duration of delivery, and coincident release of endotoxin) that give rise to variants of this syndrome.
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Supported by NIH grants NHLBI R01 0033-11-00 (F. B. Taylor, Jr.), NIAID1R29 A1-34530 (V. L. Tesh), and NIAID R01 5R01DK52083 (R. Siegler) and a Steven Lund Gift (R. Siegler).
Accepted for publication January 21, 1999.
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on human vascular endothelial cells in vitro. Infect Immun 1991, 59:4173-4179 stimulated production by cultured human proximal tubular epithelial cells. Kidney Int 1992, 42:383-389[Medline]
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