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*
Department of Pediatrics, Division of Nephrology and Hypertension,
University of Utah, Salt Lake City, Utah
Department of Pathology, Primary Children's Medical Center, Salt Lake
City, Utah
Department of Medical Microbiology and Immunology, Texas A&M
University, Health Science Center, College Station, Texas
Cardiovascular Biology Research, Oklahoma Medical Research Foundation,
Oklahoma City, Oklahoma
Correspondence to Dr. Richard L. Siegler, Professor and Division Chief, Pediatric Nephrology and Hypertension, 50 North Medical Drive, #2B441, Salt Lake City, UT 84132. Phone: 801-581-7609; Fax: 801-581-8043; E-mail: dick.siegler{at}hsc.utah.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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concentrations
rose significantly by 6 h and then declined rapidly. Urinary and plasma
interleukin-6 concentrations rose later. Glomeruli showed reduced patency of
capillary loops, fragmented red blood cells, fibrin and platelet microthrombi,
necrosis and detachment of endothelial cells, and accumulation of flocculent
material in subendothelial spaces. Damage to tubular epithelium and
peritubular capillary endothelium also was seen. Animals that received four
divided doses of Stx-1 developed no clinical or histologic features of
hemolytic uremic syndrome. It is concluded that in the primate model, disease
expression is modulated by the rate of Stx administration, and it is
speculated that in the human, the rate of Stx absorption from the gut is one
determinant of disease severity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Only approximately 10% of individuals with EHEC-mediated colitis progress to HUS, and those who develop HUS exhibit a wide variation in disease severity. Although there are reported differences in the virulence of Stx-producing E. coli (8), there also is a marked variation in disease severity in families in which the colitis is caused by the same strain of E. coli (9,10). Research therefore is needed to identify factors that modulate disease expression.
We showed previously that the baboon has in the gut and the kidney GB3 receptors that are similar in location and quantity to those of the human (11) and that a single 50- to 200-ng/kg intravenous dose of purified, LPS-free Stx-1 causes thrombocytopenia, microangiopathic hemolytic anemia, and renal TMA (12). To define further this primate model and study factors that modulate disease expression, we compared the effects of a 100-ng/kg dose of Stx-1, given either rapidly as a single intravenous bolus or slowly as four 25-ng/kg doses administered at 12-h intervals. Results also were compared with those observed in control animals that received an injection of saline.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental Protocol
Baboons (Papio c. anubis or Papio c. cynocephalus) were obtained from the
breeding colony at the University of Oklahoma Health Sciences Center or from
Osage Research (Osage Beach, MO). Juvenile males were used for the Stx
experiments (Table 1) because
of the difficulty in catheterizing the urinary bladders of females. The
animals weighed between 4 and 7 kg and developmentally were comparable to 3-
to 6-year-old humans. They were in good health without evidence of
tuberculosis and were observed for 30 d before experiments.
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Solid food was withheld for 12 h before the experiments in preparation for general anesthesia, but free access to water was allowed to prevent dehydration and subsequent oliguria and prerenal azotemia. Animals were anesthetized with intramuscular ketamine (14 mg/kg) and orally intubated, and a percutaneous venous catheter was placed in a cephalic vein. They were kept in a light plane of general anesthesia with intravenous pentobarbital (2 to 3 mg/kg every 30 min, as needed) administered through the venous catheter. A warming pad was used, and body temperature was monitored via a rectal probe and monitor. A femoral artery and vein were surgically exposed with the use of aseptic technique and cannulated for BP and central venous pressure (CVP) measurements and blood sampling. The cannulas were attached to heparin locks (10 U of heparin/ml) and buried under the skin to prevent access by the animals. Arterial BP and heart rate were monitored with the use of a multichannel Hewlett Packard monitor (Palo Alto, CA); CVP was measured via a glass manometer. Ceftriaxone (50 mg/kg) was administered by intramuscular injection once every 24 h. Blood samples were obtained from the femoral venous catheter, and urine was obtained via a Foley catheter temporarily inserted through the urethral meatus.
Before the intravenous administration of Stx-1 (or 0.9% saline in the control animals), vital signs were recorded and baseline (T-0) blood and timed urine samples were collected. After the intravenous administration of Stx-1 or 0.9% saline, the animals were maintained in a light plane of anesthesia and observed on the surgical table for 2 h. The animals then were returned to their cages. Additional blood and urine samples were collected at 6 h to capture early cytokine release and then again at 12 h and every 12 h thereafter. At the beginning of each collection period, animals were weighed and vital signs were obtained. For prevention of hypovolemic (prerenal) oliguria and azotemia, 0.9% saline was administered intravenously if the weight was less than baseline (in a volume sufficient to bring weight back to baseline). One or more infusions of 10 ml/kg were given if the CVP was <3 cm of water, if the mean BP was <75 mmHg, or if the urine output was <2 ml/kg per h. Fluids were withheld, however, if the animal's CVP was >10 cm/water. Timed urine collections were started after any required intravenous fluid had been administered.
Automated blood cell counts were performed on a Technicon H-1 System (Miles, Inc., Diagnostics Division, Naperville, IL), and Wright-stained peripheral blood smears were examined microscopically. Routine blood chemistry studies were performed on a Vitros 700 Chemistry Analyzer (Johnson & Johnson Clinical Diagnostics, Inc., Rochester, NY). Urine was analyzed, with the use of reagent strips (Multistix 10 SG; Bayer, Elkhart, IN), for blood, protein, specific gravity, pH, nitrites, and leukocyte esterase. After centrifugation, the urinary sediment was examined microscopically.
Experiments usually were terminated at 72 h (sooner if the animal became moribund) by the intravenous injection of pentobarbital (100 mg/kg.). Two animals that received the toxin slowly as divided doses were allowed to survive for 192 and 312 h, respectively, to exclude the possibility of delayed onset HUS. A complete post-mortem examination was performed after death.
Tissue Preparation
Light Microscopy. Sections of cortex and medulla from the
mid-lateral aspect of both kidneys were immersed in 10% neutral buffered
formalin for at least 6 h and then dehydrated and paraffinized with the use of
an automated tissue processor (Tissue Tek; Sakura; Finetek, Torrance, CA).
Sections 3 to 4 µm thick were stained with hematoxylin and eosin and with
periodic acid-Schiff and other stains when necessary.
Electron Microscopy. Small portions of cortex from the midregion of the left kidney, obtained within 5 min of death, were placed in 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium Cacodylate with 2.4% sucrose, and 8 mM calcium chloride for at least 1 h and then processed through graded alcohols and embedded in Spur's low-viscosity embedding medium. One µm of semithin sections was stained with toluidine blue for light microscopy, and thin sections were stained with lead citrate and uranyl acetate and examined with a Hitachi H-700 transmission electron microscope (Tokyo, Japan).
Immunofluorescence Microscopy. Sections of renal cortex were flash-frozen in OCT-embedding compound, and frozen sections were stained with a fluorescein-conjugated IgG fraction of goat antihuman fibrinogen (ICN Pharmaceuticals, Aurora, OH) and examined with a fluorescence microscope.
Scoring of Glomerular Histopathology
The histologic findings in 100 consecutive glomeruli from animals in each
group (i.e., single-dosed [n = 3], split-dosed [n =
3], saline controls [n = 2]) were scored in a blinded manner as
described previously (12).
Cytokine Assays
Solid-phase enzyme-linked immunosorbent assay commercial kits (R&D
Systems, Inc., Minneapolis, MN) were used to measure cytokines in urine and
plasma.
Statistical Analyses
Because the sample sizes were small and therefore not normally distributed,
data were expressed as medians and ranges, and the nonparametric
Kruskal-Wallis test was used to determine significance. A P < 0.05
was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No clinical signs of HUS were observed in the saline control animals (n = 5) or in the animals that received the Stx as four divided doses (n = 5). One split-dosed animal that manifested no signs of clinical illness through 96 h of observation, however, was found dead at 120 h.
Laboratory Data
Cytokine Values. Urinary (pg/mg creatinine) tumor necrosis
factor- (TNF-) median values rose in the single-dosed animals
from 0 at baseline (hour 0) to 4.39 at 6 h, compared with values of 0.94 in
control animals (P = 0.024) and 1.46 in split-dosed animals
(P = 0.0495). Levels then declined rapidly
(Figure 1A).
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Interleukin-6 (IL-6) urine values (Figure 1B) in the single-dosed animals rose from 0.73 at baseline to 4.9 at 24 h, which were significantly higher than those of control (0; P = 0.043) or split-dosed animals (0.64; P = 0.045). Values continued to rise and by 48 h were 101.28, compared with 0 in controls (P = 0.013) and 1.4 in split-dosed animals (P = 0.027). Sixty-h values remained elevated (259) relative to control values (0; P = 0.028) and split-dosed animal values (0.79; P = 0.0495). Insufficient numbers of single-dosed animals survived to 72 h to allow meaningful analysis.
Plasma TNF- concentrations generally were undetectable.
Plasma IL-6 concentrations (pg/ml), however, rose in the single-dosed group from 9.3 at baseline (Figure 1C) to 72.94 by 36 h, which was significantly higher (P = 0.011) than that of control animals (18.1). By 48 h, the median value in the single-dosed group was 102.4, compared with 18.3 (P = 0.028) and 19.6 (P = 0.018) in control and divided-dosed animals, respectively. By 60 h, it had risen further to 132.6, a value that did not achieve statistical significance when compared with controls (10.6; P = 0.05) or split-dosed animals (17.4; P = 0.077).
Urinalysis. Granular casts (Figure 2A) were abundant in all but one of the single-dosed animals by 24 h and occasionally were noted as early as 12 h after Stx-1 infusion. Casts were infrequent in the split-dosed group and were absent in the control group. Proteinuria appeared within 24 h in the animals that received the single dose of toxin and became pronounced (i.e., 4+) by 36 h (Figure 2B); hematuria began by 36 h and was prominent by 48 h (Figure 2C). Hematuria and proteinuria in the split-dosed and control groups were only intermittent and modest in amount, with the exception of the split-dosed animal that died at 120 h; this animal had 4+ proteinuria at 48 h that decreased to 1+ by 60 h.
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Renal Function. Acute renal failure began approximately 24 h after the onset of proteinuria, i.e., by 48 h, in the animals that received the single intravenous bolus dose of Stx-1. The blood urea nitrogen and serum creatinine concentrations are depicted in Figure 3. The blood urea nitrogen (median) in the single-dosed group at 24 h was 10.5 mg/dl, which was significantly greater (P = 0.017) than that of control animals (7 mg/dl). By 36 h, it was 15.5 mg/dl, compared with 8 mg/dl in the divided-dosed animals (P = 0.006). The serum creatinine concentration was significantly greater (P = 0.011) than that of control animals at 36 h (0.65 mg/dl versus 0.5 mg/dl). By 48 h, it was significantly higher than that of divided-dosed animals (0.75 mg/dl versus 0.6 mg/dl; P = 0.021).
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Platelets. Animals that received the 100 ng/kg Stx-1 as a single intravenous bolus dose developed progressive thrombocytopenia. By 36 h, the platelet count was 126.5 x 109/L compared with 217 x 109/L (P = 0.01) in the control group and 171 x 109/L (P = 0.028) in the split-dosed animals (Figure 4A). Thrombocytopenia was severe (23.5 x 109/L; P = 0.032) by 60 h. Animals in the divided-dosed group showed only a modest and transient decrease in platelet count (median nadir, 156 x 109/L) that was not significantly different from that of controls.
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Red Blood Cells. Schistocytes (Figure 4B) in the single-dosed group were significantly more numerous than in the control (P = 0.044) and split-dosed (P = 0.045) groups by 36 h. They were abundant (median, 30.0%) by 60 h. Fragmented cells were only occasionally seen in the divided-dosed or control groups. Although hematocrit values (not shown) fell in all animals as a result of multiple blood sampling, the median hematocrit value at 60 h in the single-dosed group was 27.3% compared with 34.4% in the control animals and 31.7% in the animals that received divided doses of Stx. These differences did not achieve statistical significance.
Necropsy Findings
Five of the six animals that received the single intravenous dose of Stx-1
underwent necropsy immediately after death; in one, necropsy was performed
approximately 8 h after death. The saline control animals and four of the five
that received the divided doses of Stx-1 underwent necropsy immediately after
the administration of the euthanizing agent. Necropsy was delayed
approximately 72 h in the split-dosed animal that died unexpectedly at 120
h.
No renal TMA or other significant gross, histologic, or ultrastructural abnormalities were seen in control animals or in those that received divided doses of Stx-1, except for the split-dosed animal that died at 120 h. This animal had marked renal medullary congestion and patchy hemorrhage plus extensive tubular injury but no TMA. The lungs also showed diffuse congestion, patchy edema, and focal hemorrhage, but no significant gastrointestinal, cardiac, or brain abnormalities were found.
The kidneys of the animals that received the Stx-1 as a single intravenous bolus showed congestion and/or hemorrhage at the corticomedullary junction by gross examination. The median indexed weight of the kidneys (kidney weight/body weight x 100) was 0.88 compared with 0.54 (P = 0.011) and 0.52 (P = 0.025) in the control and split-dosed groups,1 respectively. The adrenal glands were congested or hemorrhagic in five of the six animals that received Stx as a single intravenous bolus. The small and, more notably, the large intestines in four of these six showed mucosal congestion. Two had pulmonary hemorrhages, and all exhibited myocardial petechiae. Fluid that had the appearance of transudate was found in the peritoneal, pericardial, and, occasionally, the pleural spaces. Mild to moderate cerebral edema was noted in three, but no evidence of hemorrhage or infarction was found on gross examination of the intact brain or the cut sections.
Histologically, glomeruli from animals that received Stx as a single intravenous bolus showed reduced patency of capillary lumens and fragmentation of red blood cells (Figure 5C), and abundant fibrin could be demonstrated by immunofluorescence microscopy (Figure 5D). Electron microscopy revealed platelet and fibrin thrombi within lumens, necrosis and detachment of endothelial cells, and accumulation of finely granular electron lucent material in subendothelial spaces (Figure 6B). Renal tissue from animals that received four divided doses of Stx (Figures 5, A and B, and 6A) showed no abnormalities. Scoring of 100 consecutive glomeruli in one kidney from the animals in the single-dosed, split-dosed, and control groups (Table 3) revealed that approximately 50% of the glomeruli in the single-dosed group showed evidence of TMA in the form of reduced patency of capillary loops, fragmented red blood cells, and/or fibrin thrombi, compared with 1% or less in the split-dosed and control groups, respectively.
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Renal tubules in the single-dosed group showed vacuolation, apical blebbing, and detachment by light (Figure 5C) and electron microscopy (Figure 7A). Peritubular capillaries showed damage to the endothelial cells and adherent platelets (Figure 7B).
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None of the brains showed hemorrhage, infarction, herniation, or large vessel thrombi; light and immunofluorescence revealed no intravascular fibrin/thrombi. Toluidine blue-stained 0.5-µ sections, however, showed variable expansion of cortical perivascular spaces (edema) that was more prominent in the animals that had had seizures, and transmission electron microscopy showed endothelial swelling and vacuolation in some of the vessels. No consistent differences were noted between groups, including those that experienced seizures, however.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The baboon's response to a single 100-ng/kg intravenous bolus infusion of purified LPS-free Stx-1 is similar to that seen in children whose HUS follows Stx-producing E. coli enteric infection. That is, both the baboon and the human develop severe thrombocytopenia, microangiopathic hemolytic anemia, and an acute nephropathy characterized histologically by glomerular endothelial cell injury and fibrin-platelet microthrombi.
The split-dosed and control animals did not develop HUS and experienced in their platelet counts only a mild, nonprogressive decrease that probably was due to platelet activation secondary to the surgery and placement of indwelling femoral vessel catheters. They had no schistocytosis or renal TMA. The death of one of the five split-dosed animals at 120 h was unexpected because there had been no progressive thrombocytopenia, schistocytosis, or azotemia throughout the 60 h of blood sampling and no clinical signs of illness throughout 96 h of observation. The animal did exhibit 4+ proteinuria without casts or hematuria at 48 h, but it had decreased to 1+ by 60 h. Although the precise cause of death could not be determined by necropsy, there was marked pulmonary congestion and severe renal medullary congestion and tubular damage but no TMA.
The animal's clinical and histologic response to the rapid administration
of the 100-ng/kg dose was associated with an early transient rise in urinary
but not plasma, TNF- concentrations. This observation suggests that the
source of the TNF was the kidney. Stx specifically induces TNF production in
the kidney of transgenic mice that have a chloramphenicol acetyl-transferase
reporter gene that is coupled to a TNF promoter, but not in other tissues
(15). Moreover, it induces
release of TNF and mRNA when incubated with cultured human proximal renal
tubular cells (16).
TNF- has many biologic properties that could be important in the
pathogenesis of HUS and is elevated in the urine
(17,18)
of children during the acute phase of HUS. TNF acts on endothelial cells to
induce procoagulant activity
(19) and to promote white
blood cell adhesiveness, probably through increased production of soluble
intracellular adhesion molecule
(20). It also induces the
release of von Willebrand factor
(21), which facilitates
formation of platelet thrombi and increases the sensitivity of cultured human
glomerular endothelial cells to the cytotoxic effects of Stx-1
(22). It also may play a role
in triggering programmed cell death, in that it induces apoptosis in cultured
bovine glomerular endothelial cells
(23). Even so, it is not yet
known whether TNF- is required for disease expression, only augments
the pathogenic cascade, or is merely an epiphenomenon.
We also found a progressive increase in both urine and plasma IL-6
concentrations in the animals that received the toxin as a single dose. Levels
became pronounced approximately 48 h after Stx-1 administration and continued
to increase until death. IL-6 is a cytokine that, like TNF-, has
prothrombotic properties (24).
It is produced by macrophages and mesangial, endothelial, and other cells and
is elevated in the urine and plasma of children with HUS
(18). Its role in the
pathogenic cascade is unknown, however, and it could be merely a marker of
disease activity.
Three of the six that received the 100-ng/kg Stx-1 as a single dose developed seizures, not an uncommon occurrence in children with HUS (25). The seizures were not associated with hypertension or marked abnormalities in the serum concentration of glucose, calcium, or electrolytes; azotemia was no more severe in those that seized than in those that did not. Although children with HUS-related seizures generally have more severe electrolyte abnormalities and azotemia than those without convulsions (26), that often is not the case and illustrates our limited understanding of HUS encephalopathy. Two of the three baboons with seizures were found by gross examination to have mild to moderate cerebral edema at necropsy and electron microscopy showed perivascular edema. It seemed to have been part of a more generalized capillary leak disorder, because the majority of animals had fluid in the pleural, pericardial, and/or peritoneal spaces that had the appearance of a transudate. Even though we were unable to determine whether the cerebral edema played a role in the pathogenesis of the convulsions, it is notable that severe generalized cerebral edema and associated convulsions are frequent causes of death in children with HUS (26).
Intestinal involvement was limited to mucosal congestion. We showed previously, however, that the baboon (and human) have GB3 receptors in the large intestine and that when larger amounts (2000 ng/kg) of Stx-1 are given intravenously to the baboon, severe gut lesions occur (12). We speculate that Stx given intravenously in this model is preferentially bound to GB3 receptors in the kidney. When toxin is given in amounts sufficient to saturate renal GB3 receptors, however, Stx-mediated damage also is seen in the gut. Intestinal damage may be severe in the human model because the bacterial colitis facilitates absorption of Stx directly into the gut microcirculation.
The variation in the incidence and severity of HUS after EHEC colitis is not well understood. Although the subcutaneous injection of small amounts of either Stx-1 or Stx-2 are approximately of equal potency in causing HUS in the greyhound model (B. W. Fenwick, Department of Pathology and Microbiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, personal communication, December 1999), this may not be true in the human. There are epidemiologic data suggesting that enteric infection with Stx-2—producing E. coli is more likely to lead to HUS (27) than is infection with bacteria that produce only Stx-1. Moreover, Stx-2 is markedly more cytotoxic to cultured human glomerular endothelial cells than is Stx-1 (28). Therefore, although Stx-1 and Stx-2 both interrupt protein synthesis in a similar manner, Stx-2 seems to be more efficient in doing so. We elected to use Stx-1 because it is easier to purify and was available in our laboratory. We recognize that smaller amounts of Stx-2 probably would elicit the same biologic response. Studies to compare the baboon's response to both Shiga toxins are planned.
It is common for EHEC colitis to affect multiple family members, yet only a minority develop HUS. Moreover, there is often wide variation in disease severity in those family members who progress to HUS. For example, we treated 9 families in whom postdiarrheal HUS developed concurrently in two family members, presumably after exposure to the same strain of Stx-producing E. coli (9). The severity of the HUS differed markedly between members in approximately one half of families; severe dialysis-dependent renal failure (and occasionally death) occurred in one member, but only mild disease occurred in the other.
Although numerous factors probably modulate the clinical response to Stx, our observations suggest that both the amount and the rate of toxin absorption are important. We showed previously that single doses as low as 50 ng/kg of Stx-1, having a specific cytotoxicity of 2 x 106 CD50/µg protein, predictably causes severe HUS (12). In the present study, we found that a dose of 25 ng/kg does not result in HUS. The threshold for HUS after a single rapidly administered dose of Stx-1, therefore, seems to be between 25 and 50 ng/kg. Moreover, a dose (e.g., 100 ng/kg) that produces severe HUS when administered rapidly does not when given slowly. In humans, there may be a threshold, relative to both quantity and rate of Stx absorption from the gut, that must be reached for HUS to develop.
Our primate model observations may have therapeutic implications relative to Stx-mediated HUS in humans. Strategies designed to block or slow the rate of Stx absorption from the gut, such as the oral administration Synsorb Pk (Sysorb Biotech, Inc., Calgary, Alberta, Canada) (29), should be helpful.
On the basis of cell culture studies (30) and experience with a mouse model (31), there is evidence that both Stx and LPS are important in the pathogenesis of postdiarrheal HUS. Although that may be true, LPS is not required in our primate model. We speculate that a sufficient dose of Stx alone is able to cause HUS in humans as well. That is not to say, however, that LPS might not amplify the response to Stx, and primate experiments are under way to study the role of LPS in Stx-mediated disease.
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Acknowledgments |
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The authors gratefully acknowledge Andrew T. Pavia, M.D., Randall Lou, M.D., Nathaniel D. Denkers, Brett D. Welch, Mary Ann Harmon, the personnel of the University of Utah's Animal Resource Center, and Jana Johnson, who assisted with the experiments, data collection, statistical analysis, and/or preparation of this manuscript.
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Footnotes |
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1 The split-dosed animal that died unexpectedly and had renal
hemorrhage/congestion and tubular injury but no TMA was excluded from
analysis. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Inflammation18
: 163-173,1994[Medline]
on verocytotoxin cytotoxicity in purified human glomerular
microvascular endothelial cells. Kidney Int51
: 1245-1256,1997[Medline]
and lipopolysaccharide induce apoptotic cell death in bovine
glomerular endothelial cells. Kidney Int55
: 2322-2337,1999[Medline]
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single dose of Stx; [UNK], split dose of Stx; 
, controls; *,
significance (P < 0.05) between single- and split-dosed groups;
**, significance between single-dosed and control groups.
