Background

Both verotoxin (VT) 1 and VT2 share the same receptor, globotriaosyl ceramide (Gb₃). Although VT1 is slightly more cytotoxic in vitro and binds Gb₃ with higher affinity, VT2 is more toxic in mice and may be associated with greater pathology in human infections. In this study we have compared the biodistribution of iodine 125 (¹²⁵I)-VT1 and ¹²⁵I-VT2 versus pathology in the mouse.

Methods

¹²⁵I-VT1 whole-body autoradiography defined the tissues targeted. VT1 and VT2 tissue distribution, clearance, and tissue binding sites were compared. The effect of a soluble receptor analogue, adamantylGb₃, on VT2/Gb₃ binding and in vivo pathology was assessed.

Results

¹²⁵I-VT1 autoradiography identified the lungs and nasal turbinates as major, previously unrecognized, targets, while kidney cortex and the bone marrow of the spine, long bones, and ribs were also significant targets. VT2 did not target the lung, but accumulated in the kidney to a greater extent than VT1. The serum half-life of VT1 was 2.7 minutes with 90% clearance at 5 minutes, while that of VT2 was 3.9 minutes with only 40% clearance at 5 minutes. The extensive binding of VT1, but not VT2, within the lung correlated with induced lung disease. Extensive hemorrhage into alveoli, edema, alveolitis and neutrophil margination was seen only after VT1 treatment. VT1 targeted lung capillary endothelial cells. Identical tissue binding sites (subsets of proximal/distal tubules and collecting ducts) for VT1 and VT2 were detected by toxin overlay of serial frozen kidney sections. Glucosuria was found to be a new marker of VT1- and VT2-induced renal pathology and positive predictor of outcome in the mouse, consistent with VT-staining of proximal tubules. Lung Gb₃ migrated on thin-layer chromatography (TLC) faster than kidney Gb₃, suggesting a different lipid composition. AdamantylGb₃, a soluble Gb₃ analogue, competed effectively for Gb₃ binding by VT1 and VT2 in vitro. However, the effect in the mouse model (only measured against VT2, due to the lower LD₅₀, a concentration required for 50% lethality) was to increase, rather than reduce, pathology and further reduce the VT2 serum clearance rate. Additional renal pathology was seen in VT2 + adamantylGb₃-treated mice.

Conclusions

The lung is a preferential (Gb₃) "sink" for VT1, which explains the relatively slower clearance of VT2 and subsequent increased VT2 renal targeting and VT2 mortality in this animal model.

METHODS

Reagents

Monoclonal antibody (mAb) PH1, specific for the VT1 B subunit, has been previously described⁶². PH1 was purified from culture supernatant using Protein A-sepharose chromatography. Rabbit polyclonal antiserum against the purified VT1 B subunit was prepared, as previously described⁶³. Rabbit antiserum raised against VT2e and reactive with VT2 was a generous gift of Dr. Carlton Gyles, University of Guelph, Guelph, Canada. Antibody against the facilitative glucose transporter GLUT-2, selectively expressed in the S1 segment of proximal tubules^64,65, was obtained from Alpha Diagnostic International, Inc. (San Antonio, TX, USA). Biotinylated Dolichos biflorus agglutinin (which binds -N-acetyl galactosamine, highly expressed in collecting ducts⁶⁶) and peanut agglutinin (which binds terminal -galactose and galactosyl (-1,3) N-acetylgalactosamine; distal tubules) were purchased from Vector Laboratories (Burlingame, CA, USA).

Toxins

VT1 was purified from strain JB28⁶⁷, as previously described⁶⁸. VT2 was purified from E. coli R82pJES120DH5a, kindly provided by Dr. J. Samuel (Texas A&M University, College Station, TX, USA). Nucro-Technics, Inc. (Toronto, Ontario, Canada) analyzed purified toxins for endotoxin using the Limulus amoebocyte lysate method. VT1 had <0.1 EU/g (<10 pg/g) while VT2 had <12.5 EU/g (<1.25 ng/g). These lipopolysaccharide (LPS) levels are far below those shown to cause synergistic or protective effects on VT2 pathology⁶⁹.

Purified toxins were labeled with iodine 125 (¹²⁵I) using iodogen, as described previously⁷⁰. The protein concentration of the isolated iodinated VTs was determined using the bicinchoninic acid (BCA) protein assay (Pierce Chemical Co., Rockford, IL, USA). Greater than 97% of recovered radioactive material was protein-bound as determined by trichloroacetic acid precipitation. Specific activities were typically 8 to 12 10⁶ cpm/g for VT1 and VT2. To verify that radiolabeling of the toxins did not affect activity, a Vero cell cytotoxicity assay was performed to compare labeled and unlabeled toxin. Iodinated VT1 was heat-inactivated by treatment at 90 to 100°C for 30 minutes. Binding of denatured toxin to Vero cells was reduced by>90% compared to native ¹²⁵I-VT1.

Mice

The mice were 12- to 16-week-old female BALB/c mice (weight range, 20 to 30 grams) that were purchased from Charles River, Canada. Male mice were used in the whole-body autoradiography studies. The mice were maintained under a 12-hour light-dark cycle and fed with standard diet and water ad libitum.

Biodistribution

Whole-body autoradiography.

Whole-body autoradiography was performed by Oread Biosafety, Inc. (Metabolism and Pharmacokinetics Division, Lawrence, KS, USA). Mice were pretreated with oral potassium iodide 24 hours prior to injection of ¹²⁵I-VT1 to block thyroid uptake of any free ¹²⁵I. ¹²⁵I-VT1 (1.12 g) was administered intravenously through the tail vein and the animals were sacrificed by CO₂ inhalation 15 minutes (N = 2) or 45 minutes (N = 3) later. Animals were frozen in hexane/dry ice and embedded in carboxymethyl cellulose. Sagittal cryosections (40 M) using a Richert-Jung Polycut S cryomictrotome (Leica Microsystems, Inc., Bannockburn, IL, USA) were collected on adhesive tape, dried, and exposed to Kodak SB-5 x-ray film. Hematoxylin and eosin (H&E) stained sections used for autoradiography were used for tissue assignment.

Quantitative tissue distribution.

Mice were injected via the tail vein with 2 g/kg ¹²⁵I-VT1 or ¹²⁵I-VT2 (about 50 ng/mouse) diluted in 200 L saline. In these studies, no allowance for the difference in LD₅₀ values for these toxins was made and pathologic-induced changes for VT2 might have occurred. Pilot experiments were performed to examine toxin distribution at 1, 2, or 8 hours post injection and the effects of perfusion prior to tissue collection on the biodistribution data obtained. For final clearance/biodistribution studies, groups of mice (N = 8) were injected with toxin and a 50 L blood sample was obtained from the lateral saphenous vein at 5 and 30 minutes post injection. At 60 minutes post injection, mice were anesthetized (0.1 mL/g 3.6% chloral hydrate intraperitoneally) and a 200 L blood sample was obtained from the orbital plexus. Upon taking the last blood sample, mice were sacrificed immediately by cervical dislocation and tissues were removed for analyses of radioactivity using a gamma counter.

Effect of adamantylGb₃ on VT2 biodistribution

The semi-synthetic, soluble Gb₃ derivative, N-adamantylGb₃ (adaGb₃), was prepared from human renal Gb_3, as previously described⁶¹. Two groups of mice (N = 8) were injected intravenously with a mixture of 4 mg/kg adaGb₃ and 2 g/kg ¹²⁵I-VT2 (representing about 100 g (100 nmol) of adaGb₃ and 50 ng VT2 (0.7 pmol) per mouse) in 200 L. The procedures followed matched those used in the biodistribution studies discussed above.

Clinicopathologic studies with VT2 challenged mice

The LD₅₀ of VT2 in mice is 400-fold lower than that of VT1⁴⁶. Therefore, VT2 was chosen for in vivo protection studies using adaGb₃. Initial studies showed that a dose of 2 ng/mouse (80 ng/kg) caused development of fatal disease in 100% of mice 80 to 105 hours post-injection. This dose represents 1.5 to 4 times the reported LD₅₀ in Balb/c mice^69,71.

The ability of adaGb₃ to alter the VT2 disease course with respect to clinical, biochemical, and histologic symptoms was investigated. In all cases, mice were injected intravenously through the tail vein (N = 5) for each group. Group 1 received 2 ng (80 ng/kg) VT2. Group 2 received a mixture of 2 ng VT2 and 100 g (4 mg/kg) adaGb₃. Group 3 was injected intravenously with 4 mg/kg adaGb₃ alone. A fourth group of mice (N = 3) was injected intravenously with sterile saline alone. AdaGb₃-VT2 mixtures were pre-incubated for 20 to 30 minutes at room temperature prior to injection.

In experiments involving blood collection, 24 hours prior to injection, a 200 L blood sample was taken from the lateral saphenous vein. At 72 hours post injection, mice were anesthetized with chloral hydrate (3.6% 0.01 mL/g intraperitoneally) and a 300 L blood sample was taken from the orbital plexus. After obtaining the second blood sample, mice were immediately sacrificed by cervical dislocation and tissues were processed as described under Histopathology below.

All blood samples were collected with heparinized capillary tubes. Blood was centrifuged immediately upon collection, plasma and cells were separated, and plasma was stored at –20°C until analysis. To monitor for signs of renal dysfunction, plasma samples were analyzed for blood urea nitrogen (BUN) and creatinine at the Department of Pediatric Laboratory Medicine, Hospital for Sick Children, in Toronto, Ontario, Canada. Blood glucose levels were measured using a One Touch Basic blood glucose monitoring system (Lifescan Canada, Ltd., Burnaby, B.C., Canada). Urine samples were collected upon spontaneous voiding. Using Albustix (Bayer, Inc., Toronto, Ontario, Canada), urine was analyzed for glucose, protein, pH, specific gravity, erythrocytes, and leukocytes.

Histopathology

Animals treated with VT1, VT2, VT2 + adaGb₃, adaGb₃, or saline were sacrificed by cervical dislocation. Kidney, lung, brain, spleen, and liver were fixed in neutral-buffered formalin and then paraffin-embedded and sectioned. All sections were stained with H&E and, in addition, kidney sections were stained with periodic acid-Schiff (PAS) to identify the brush-border of proximal tubules.

VT staining of frozen kidney sections

A fresh tissue sample was placed in Tissue-Tek O.C.T. compound (Miles, Inc., Diagnostics Division, Elkhart, IN, USA) and snap-frozen in liquid nitrogen. Serial 6 m frozen sections were cut and stored at –70°C. The immunoperoxidase VT staining was performed, as described⁷². Sections were dried overnight and then blocked separately for endogenous peroxidase, avidin, and biotin. The sections were incubated with 50 ng/mL of VT1 or VT2 diluted in 1% goat serum/phosphate-buffered saline (PBS) for 30 minutes. After washing, polyclonal anti-VT1 or anti-VT2 was added and bound antibody was detected with a biotinylated goat anti-rabbit IgG-steptavidin peroxidase system. Sections were counterstained with hematoxylin, dehydrated, and mounted. Serial sections were also stained with PAS, anti-GLUT-2, biotinylated Dolichos biflorus agglutinin, peanut agglutinin, or 1% goat serum/PBS as antibody control.

In situ immunodetection of targeted VT

Mice were injected through the tail vein with 10, 50 or 100 g of VT1 or VT2 in 200 L of saline. After 1 hour, the animals were sacrificed by cervical dislocation and organs were collected and divided. Half was placed in O.C.T compound and snap-frozen in liquid nitrogen for preparation of frozen sections. The remainder was placed in neutral buffered formalin for a minimum of 16 hours before being paraffin-embedded and sectioned.

VT was detected in the frozen sections essentially as described above for VT staining of frozen kidney sections, with the following two exceptions. The frozen sections were fixed with 100% acetone for 10 minutes prior to assay rather then dried overnight, and the VT1 binding step was omitted. Tissues from mice injected with saline rather than VT served as controls for non-specific binding of anti-toxin antibodies.

Paraffin sections were de-paraffinized by immersion in xylene, followed by baths of decreasing ethanol content (100%, 95%, 70% ethanol in water), and finally, water. After de-waxing, antigen retrieval was required for detection of VT in kidney sections. Antigen unmasking was performed by heating the de-waxed sections in 0.01 mol/L citrate buffer pH 6, in a pressure cooker⁷³.

VT TLC overlay

Glycosphingolipids (GSLs) were extracted from lungs and kidneys (pooled from 5 animals) as previously described³⁹ and equivalent aliquots according to tissue weight were separated by TLC and overlaid with either VT1 or VT2⁷⁴. Toxin binding was visualized using ECL detection (Amersham Biosciences, Piscataway, NJ, USA).

Statistical analysis

Results are expressed as the mean standard deviation.

RESULTS

Whole-body autoradiography after ¹²⁵I-VT1 administration.

Several illustrative sagittal sections are shown in Figure 1. No additional organs were labeled in other sections. VT1 labeling of the kidney cortex, lung, and the nasal turbinates were the most prominent features of the autoradiogram Figure 1. Labeling of the bone marrow of the spine, long bones, and ribs was also extensive. The labeling in the lung could be seen to be non-uniform since bronchioles were not labeled and foci within the lung could be distinguished on less exposure (Figure 1, lower panel). The spleen was significantly labeled, but less than lung. The liver was barely labeled above the background. No labeling of the gastrointestinal tract, central nervous system, brain stem, spinal chord, testes, lymph nodes, thymus, reproductive tract, pancreas, blood, skeletal/cardiac muscle, dermis, or epidermis was seen.

Figure 1.

Autoradiography of ¹²⁵I-VT1 injected mouse. Sample sagittal sections from several mice are shown with labeled tissues indicated. The lower panel highlights the non-uniform labeling in the lung seen on reduced exposure.

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Tissue distribution of ¹²⁵I-VT1 and ¹²⁵I-VT2

Clearance.

Blood samples were taken at three different time points. Because blood was not collected before 5 minutes, the data for zero time points were calculated from the estimated blood volume for each mouse⁷⁵ and the counts injected. To compare clearances of both VT1 and VT2, the amount of toxin as measured in the blood samples was expressed as a percentage of injected counts recovered in 50 L blood Figure 2. Within 5 minutes, 90% of injected VT1 but only 40% of injected VT2 was cleared. After 60 minutes there were sixfold less counts circulating for VT1 than for VT2. The estimated half-lives for VT1 and VT2 were 2.7 and 3.9 minutes, respectively, based on the decrease in counts between time points 0 and 5 minutes.

Figure 2.

Serum clearance of ¹²⁵I-VT1 and ¹²⁵I-VT2. Approximately 50 ng ¹²⁵I-labeled toxin was injected into mice intravenously (N = 8) and counts remaining in circulation over the next hour were measured. The effect of mixing the semi-synthetic soluble Gb₃ derivative N-adamantylGB₃ (adaGb₃) with ¹²⁵I-VT2 prior to injection was also determined. (), ¹²⁵I-VT1; (), ¹²⁵I-VT2; (), ¹²⁵I-VT2 + adaGb₃.

Full figure and legend (11K)

Biodistribution.

All measurements were at 1 hour post injection. Both groups of mice injected with ¹²⁵I-VT1 or VT2 showed tissue localization of the toxins primarily in the lungs and nasal turbinates Figure 3 consistent with the autoradiography study Figure 1. Toxin targeting to these organs has not been previously reported. However, VT1 showed a 10-fold higher lung targeting than VT2. VT2 preferentially targeted kidneys (2.7-fold that of VT1), whereas VT1 spleen targeting was twofold that of VT2. Heat inactivation, for the most part, eliminated tissue targetting Figure 3. Increased levels of the inativated VT1 in the liver and kidney probably reflect clearance of the denatured protein.

Figure 3.

Biodistribution of ¹²⁵I-VT1 and ¹²⁵I-VT2. Approximately 50 ng ¹²⁵I-labeled toxin was injected into mice intravenously (N = 8). After 1 hour, the toxin distribution to various tissues was determined and expressed as the percentage of injected counts per minute recovered per mg of tissue. (), heat-denatured VT1; (), VT2; (), VT1.

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The biodistribution of ¹²⁵I-VT1 was also compared in male and female outbred CD1 mice. Pulmonary and renal VT1 accumulation was 40% higher in male than female animals, consistent with the greater (renal) Gb₃ content in males⁷⁶, but otherwise, distribution was not significantly different from BALB/c mice (data not shown).

VT1 and VT2 immunolocalization

Kidney.

VT1 and VT2 were immunolocalized in fixed/paraffin-embedded or frozen renal sections from mice 1 hour after 10, 50 or 100 g of VT1 or VT2 had been injected Figure 4. The VT1 and VT2 immunostaining patterns were similar. Immunostaining was clear in the frozen sections, even at the 10 g dose, but morphology was better preserved in the fixed, embedded sample. Clear staining of distinct tubule subpopulations was seen for both toxins. No glomerular staining was seen (Figure 4 a to c). In the paraffin sections, it was clear that VT-targeted tubules were dilated and the epithelial cells flattened, even after this short period (Figure 4 c, d). This effect was dose dependent. Immunodetection of targeted VT1 in the unmasked paraffin sections was adequate at 100 g but poor at 50 g (and not detectable at 10 g). Nevertheless, the dismorphic tubules could be identified in the H&E section and the corresponding stained tubule identified (for the 50 and 100 g dosage). Dilated tubules were less apparent in H&E stained sections at the 10 g dosage (not shown).

Figure 4.

Immunolocalization of kidney-targeted VT1 and VT2. Frozen (A, B) or fixed, paraffin-embedded (C, D) sections of kidney were prepared from mice 1 hour after injection with either 50 g VT1 or VT2 and stained with antiserum against VT1 (A, C) or VT2 (B). A similar subpopulation of tubules are labeled by each toxin but neither bound to glomeruli (arrows). In fixed sections, tubules targeted by VT1 (C) were identified in hematoxylin and eosin (H&E)-stained serial sections as dilated and containing flattened epithelial cells (D).

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By overlay of (untreated) mouse kidney frozen sections, both VT1 and VT2 were found to bind exactly the same tubular structures Figure 5. Neither toxin bound to glomeruli (Figure 5 a, b). Labeling serial sections with the lectin D. biflorus⁶⁶ identified some of the renal structures labeled by both VT1 and VT2 as collecting ducts Figure 5e, while immunostaining with anti-GLUT-2⁶⁴ identified others as proximal convoluted tubules Figure 5f. Some distal tubules labeled with peanut agglutinin⁷⁷ were labeled with VT1 or VT2, but most were not Figure 5g.

Figure 5.

Comparison of VT1 and VT2 binding sites in frozen kidney sections. Serial frozen sections of kidney were stained with VT1 (a, c) or VT2 (b, d), the lectin Dolichos biflorus (e), anti-GLUT2 (f), peanut agglutinin (g) and periodic acid-Schiff (PAS; h). Both toxins label the same collecting ducts and proximal tubules. Comparison staining of the equivalent areas is shown (c to h). A representative glomerulus unstained by VT1 or VT2 is shown (a and b). Four collecting ducts labeled with D. biflorus and VT1, VT2 are indicated (e). The arrowhead indicates a D. biflorus-reactive collecting duct, not labeled with peanut agglutinin (g). Proximal tubules reactive with VT1, VT2 and anti-GLUT2 are indicated (f). Anti-VT2 renal staining after in vivo VT2 administration (j) is compared to PNA staining (i) of serial sections. A VT2-negative, PNA-positive distal tubule is marked * (i), while a PNA-negative, VT2-positive proximal tubule is marked * (j). Distal tubules labeled by both PNA and VT2 are indicated by arrows.

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Lung.

Frozen-section immunostaining of VT1 accumulated in the lung following intravenous injection shows areas of some intra-alveolar capillaries, and patches of pneumocytes within the alveoli are targeted Figure 6. Distinct, sometimes discontinuous, subpopulations of endothelial cells within some capillaries were labeled. Epithelial cells were, for the most part, VT1-negative. VT could not be detected in the paraffin sections perhaps due to a failure to unmask the antigen in this tissue with the conditions used. Gb₃ was detected in untreated mouse lung frozen sections by VT1 overlay, but the morphology was not sufficiently retained to allow identification of cell targets.

Figure 6.

Immunolocalization of lung-targeted VT1. Frozen sections of lung were prepared from mice 1 hour after injection with 50 g VT1. (A), Hematoxylin and eosin (H&E) stain; (B), corresponding serial section stained with anti-VT1; (C), anti-VT1 immunostaining; (D), lung section corresponding to A and B stained with non-immune serum. Discontinuous staining of endothelial cells lining a capillary is indicated in (B). Continuous staining of endothelial cells within a capillary is shown in (C). Some staining of pneumocytes is also evident.

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Pathology

Kidney.

Mice injected with 1.5 to 4 LD₅₀ (VT1 or VT2) showed kidney pathology, characteristic of tubular necrosis as previously reported⁴⁶ by day 3. Glomerular morphology appeared normal. A significant subpopulation of proximal and especially distal tubules were dilated in both VT1- and VT2-treated mice. Medullary tubular dilation was more widespread after VT1 treatment. Interstitial hemorrhage was seen for all VT-treated mice. Neutrophil migration to sites of tubular injury was not seen. Apoptosis, as monitored by terminal deoxy transferase uridine triphosphate nick end-labeling (TUNEL) staining, was not increased in the kidneys of VT-treated mice (not shown). Glucose was detected in the urine of VT-treated mice after 3 to 4 days, before the onset of any visible symptoms Table 1 and was present until the time of death. Glucosuria was an excellent predictor of mortality Table 1. Co-administration of the neutralizing anti-VT1 mAb PH1⁶² with VT1 completely protected mice from pathology and development of glucosuria Table 2. VT-treated mice did not become anuric, but did develop a urine-concentrating defect Table 1. Using urinalysis sticks, no changes in urinary protein concentration, pH, or specific gravity was measured. but erythrocytes and leukocytes were sometimes detected in urine of moribund mice.

Table 1 - Biochemical parameters of VT2-treated mice.

Full table

Table 2 - Clinical parameters of mice treated with VT1PH1 monoclonal antibodies (mAb).

Full table

Lung.

Lung sections of 2 LD₅₀⁷⁸ VT1-treated mice showed significant hemorrhage (Figure 7 a, e, j) and marked alveolitis was apparent (Figure 7 e, h). The lungs had a congested appearance Figure 7h with thickened edematous alveolar walls showing numerous entrapped erythrocytes Figure 7l. Edema within alveoli was also seen Figure 7l. The edema was often discontinuous, suggesting that some edematous material was lost during tissue preparation/staining Figure 7l. Edema of larger blood vessels was evident Figure 7k. Margination of neutrophils in venules was extensive Figure 7h and infiltration of type II pneumocytes was evident. In contrast, in VT2-treated animals, there was some slight edema and neutrophil margination evident, but lung histology was, for the most part, indistinguishable from controls (Figure 7 b, e, h compared with C, F, I).

Figure 7.

Comparison of VT1 and VT2 induced lung pathology. Mice (N = 3) were injected with 2 LD₅₀ of VT1¹⁰² or VT2, or with saline. Toxin injected mice were sacrificed when moribund and saline-treated mice at corresponding time points. Hematoxylin and eosin (H&E) staining of lung of VT1 (A), VT2 (B), and saline (C) treated mice; adjacent blood vessel and alveoli in VT1 (D), VT2 (E) and saline (F) treated mice; alveoli of VT1 (G), VT2 (H) and saline (I) treated animals. In VT1-treated mice, alveolar hemorrhage (J), blood vessel edema (K), and alveolar edema (L) were widespread.

Full figure and legend (262K)

Other.

Active acute phagocytosis was present in foci in the spleen after either VT2 or VT1 treatment. TUNEL staining revealed increased apoptosis in spleens of VT1 or VT2 but not in saline-treated mice (not shown). Also, iron pigment from increased red cell turnover was evident in the spleen of toxin-treated mice.

In the liver, lipid vacuoles were seen in VT2-treated animals but were less apparent for VT1-treated animals (not shown).

Tissue Gb₃ content

The Gb₃ content of glycolipid extracts of lung and kidney was visualized by VT1 and VT2 TLC overlay Figure 8. The mouse renal Gb₃ migrated more slowly than that from lung. VT2/Gb₃ binding was significantly less than that of VT1. A dose-dependent binding to human renal Gb₃ standard was seen, and both toxins efficiently recognized adaGb₃.

Figure 8.

VT1 /VT2 thin-layer chromatography (TLC) overlay of renal and lung glycolipids. Glycolipids were isolated from mouse lung and kidney and an aliquot representing 20 mg of tissue was separated by TLC and visualized by orcinol (A). VT binding to Gb₃ was detected by TLC overlay with either VT1 (B) or VT2 (C) as described⁷⁴. Lane 1, adaGb₃; lanes 2, 3, standard glycolipid mixture at 0.25, 0.5 g each; lane 4, mouse renal glycolipids; lane 5, mouse lung glycolipids. Glycolipid standards are, from the top of the plate, glucosylceramide, galactosylceramide, lactosylceramide, Gb₃, Gb₄, Forssman glycolipid.

Full figure and legend (105K)

Treatment with a soluble Gb₃ analog

The water-soluble Gb₃ derivative, adaGb₃⁶¹, was effective to prevent VT1 and VT2 binding to immobi lized Gb₃ as monitored by receptor enzyme-linked immunosorbent assay (RELISA) Figure 9. The IC₅₀ for VT1 was 10 mol/L⁶¹ while that for VT2 was higher (50 mol/L). Due to the much lower LD₅₀ in mice, we used VT2 to determine whether this protection in vitro could be effective in vivo. We mixed 500 mol/L adaGb₃ with approximately 1 LD₅₀ VT2 and injected it into the animals. Surprisingly, mice co-injected with VT2 + adaGb₃ were found to die more rapidly than those injected with VT2 alone (96 hours compared with 72 hours). Indeed, since a near-LD₅₀ dose was administered, in some cases, the mice treated with VT2 alone survived Table 1. Glucose was detected in the urine of VT2 + adaGb₃-treated mice earlier than for mice given VT2 alone Table 1. AdaGb₃ alone at this dosage had no obvious toxic effect in mice and renal histology was normal (Figure 10 a, d, g). In the VT2 + adaGb₃-treated mice, many tubular epithelial cells were swollen. This was seen in the cortex, but particularly in the medulla, such that many tubule lumina were occluded (Figure 10 c, f). Evidence of epithelial cell detachment from the basement membrane is seen in both VT2- and VT2 + adaGb₃-treated mice. The epithelial morphology of the affected tubules was more dysplastic, and more granular eosinophilic spheres in the tubular lumina (Figure 10 h, i), typical of acute tubular necrosis, were seen for VT2 + adaGb₃-treated mice. No brain pathology was seen for VT1-, VT2-, or VT2 + adaGb₃-treated mice.

Figure 9.

AdamantylGb₃ (adaGb₃)inhibition of VT/Gb₃ binding. VT1 or VT2 were mixed with the indicated concentration of adaGb₃ and tested for residual binding to immobilized Gb₃ in a RELISA⁶¹. (), VT1; (), VT2.

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Figure 10.

Effect of AdamantylGb₃ (adaGb₃) on VT2-induced renal pathology. Mice (N = 5) were injected intravenously with adaGb₃, VT2, or a mixture of VT2 and adaGb₃. Animals were sacrificed at 72 hours post injection and hematoxylin and eosin (H&E) stained paraffin sections of renal tissue were inspected for pathology. (A, D, G), adaGb₃-treated mice; (B, E, H), VT2-treated mice; (C, F, I), VT2 + adaGb₃-treated mice. (A to C), cortex; (D to F), medulla; (G to I), tubules at high power. Glomerular morphology appears unaffected by VT2 (B). The tubular dilation (B and E) are reversed in the VT2 + adaGb₃ mice (C, D). The epithelial cell narrowing (E and H) after VT2 treatment is also reversed, but the epithelial cells of many tubules now become enlarged such that their lumina are occluded (F). The epithelial cell layer of tubules of VT2-treated mice was often dismorphic (H) and the frequency of eosinophilic nuclei (in dying cells) increased after VT2 + adaGb₃ treatment (I, arrows)

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Comparison of the biodistribution of ¹²⁵I-VT2 with and without adaGb₃ showed that, in most tissues, VT2 tissue targeting was marginally increased, rather than decreased, in the presence of adaGb₃ (results not shown). This was consistent with the finding that the serum half-life for ¹²⁵I-VT2 was increased to 8.7 minutes in the presence of adaGb₃Figure 1.

DISCUSSION

VT-induced cell cytotoxicity^79,80 and likely in vivo pathology³⁸ are mediated by binding to its glycolipid receptor, globotriaosyl ceramide¹². Can this binding in any way explain the increased sensitivity of mice (and humans?) to VT2? The binding of VTs to Gb₃ is differentially modulated by the lipid content of the glycosphingolipid (GSL)^58,81,82 and also by the phospholipid content of the membrane in which it is contained⁸³. Due to the extensive repertoire of such modulation, Gb₃-mediated tissue targeting of these VTs subtypes could be distinct and is likely complex. Although our original studies compared the Gb₃ binding of VT1 to that of VT2c⁸², rather than VT2, VT1 and VT2 receptor binding is distinguishable⁸⁴. Although the different members of the VT family all bind Gb₃, this recognition is slightly different in each case, since removal of individual hydroxyl groups within the terminal galabiose moiety has a differential effect on the binding of each toxin variant⁷⁴.

Whole-body autoradiography has identified new tissue targets for VT1, specifically the nasal turbinates and the lung. No binding to neural, reproductive, or gastrointestinal tissue was seen. Binding within the spleen and bone marrow confirms the VT propensity for lymphoid cells^85,86,87.

Our results show a decreased clearance rate and an increased binding in kidneys for VT2 when compared to VT1, which was primarily recovered in the lungs.

Renal targeting

VT1 and VT2 have the same pattern of binding in the kidney, determined either by immunolocalization of kidney-targeted VT or by frozen-section VT overlay, both targeting proximal and distal tubules and collecting ducts. By both approaches, no labeling of glomeruli was seen. Detection of in vivo renal-targeted VT1 shows that toxin binding to tubular subpopulations has an immediate effect to dilate the targeted tubule and (as a consequence?) flatten the epithelial cells. No overt pathology in these cells could be seen.

Frozen serial section overlays showed precisely that the same renal structures were reactive with both toxins. Thus, although differences in Gb₃-mediated tissue targeting are seen with respect to the lung, Gb₃-mediated targeting in the kidney sections is not discriminatory. However, not every proximal/distal tubule was bound by the toxins, which means that there is a difference in Gb₃ expression among tubules. VT binding in the overlays was, for the most part, coincident with GLUT2, indicating binding to the S1 and S2 segment of the convoluted proximal tubule. It is in these segments of the nephron where re-uptake of filtered glucose mainly takes place. Glucose appeared in the urine approximately 3 to 4 days after VT injection and prior to the onset of any visible symptoms. Acute pancreatitis as a cause of glucosuria was excluded by showing that there was no increase of the blood glucose over time. Using Albustix to detect urinary glucose proved an easy and inexpensive way to monitor disease development in mice. Urinary glucose was seen for VT1- or VT2-treated animals and an absolute correlation with eventual death was seen. Co-administration of mAb anti-VT1 protected mice against VT1-induced mortality and development of glucosuria. VT2 mortality was exacerbated by adaGb₃, and urinary glucose was detected earlier in such mice. However, increasing VT above a lethal dosage did not increase the level of glucosuria.

The murine renal VT distribution does not correlate with the pathology observed in human pediatric HUS where mainly glomeruli are affected by the toxins^4,41,88 or with VT binding in pediatric renal sections^38,89. Glucosuria has not been reported in humans and this may prove another distinction between this model and HUS. However, HUS patients have been shown to have urinary markers of proximal tubule pathology early in the course of disease⁹⁰ and evidence of tubular cell death^91,92. Several studies have reported that cultured proximal tubule cells are highly sensitive to VT1^23,93,94. The distribution of VT in the murine kidney is similar to that seen in overlays of sections of adult human kidneys³⁸, which, on an epidemiological basis, are less susceptible to VT-induced HUS.

In situ detection of in vivo kidney-targeted VT better mimics the normal pathophysiology of systemic VT, as compared to overlays with VTs on untreated sections. In these sections, VT1 and VT2 were found in similar tubule subpopulations. This means that not only can VT bind to collecting ducts and distal and proximal tubules, it can access these structures post glomerular filtration. The fact that in situ immunostained toxins labeled tubules that were dilated and contained flattened epithelial cells indicates that VT can have an immediate effect on renal fluid balance. The cell flattening may compromise water reabsorption, resulting in local tubular dilation. The later renal pathology indicates that the tubular dilation becomes more extensive, consistent with the glucosuria, a new predictor of outcome in this animal model.

We found lipid inclusions in the liver of VT2-(3 of 3), but less in VT1-(1of 3) treated mice. This suggests that VT2 induces a more severe, generalized metabolic stress such that lipid stores are mobilized for breakdown.

Lung targeting

Some degree of pulmonary symptoms has been reported in a minor fraction of HUS patients⁹⁵. VT1, but not VT2, was detected in lung sections obtained from a patient who died of HUS²², despite the fact that both VT1 and VT2 were detected in the kidney of the same patient⁹⁶. Reported pulmonary histopathology includes microthrombosis, capillary congestion in the alveolar septa⁹⁷, and hemorrhage⁹⁸. Mechanisms responsible for this pathology are thought to be the same as mechanisms causing angiopathy at the renal level. Our biodistribution study shows that the lungs are a major target for both toxins, although 10-fold more VT1 than VT2 was bound.

When detecting injected VT1 in the lung sections, we found the toxin to be localized primarily in capillaries in some of the alveolar septa. Based on the extent of VT1-induced pathology, we expected the lung distribution to be more widespread. However, VT1-induced hemorrhage may initiate the more generalized pathology. Since VT binding is restricted to pulmonary capillaries, high local VT1 levels must accumulate to account for the biodistribution observed. Extensive localized hemorrhage and edema were evident. Margination of polymorphonuclear neutrophils was seen throughout the lung, indicating an active inflammatory response. The high level of VT1 absorbed by the lungs could explain the difference in LD₅₀ for the toxins. The lungs could function as an absorbing buffer, which permits less toxin to reach the kidneys where it could otherwise have caused more life-threatening pathology. However, we did not assess the kidneys of mice given sub-lethal doses of VT1.

A similar effect may be relevant to HUS in humans. Although the renal Gb₃ increases with age³⁹, susceptibility to VT-induced HUS is greatest in the very young when the receptor is expressed in the glomerulus³⁸. Thus, the increased expression of tubular Gb₃ as a function of age might, to some extent, prevent toxin reabsorption by providing a similar, less crucial VT "sink."

VT receptor isoforms

The Gb₃ species from kidney and lung clearly show differential migration on TLC, indicating differences within the lipid moiety, which may affect VT binding⁹⁹. Mouse renal Gb₃ has been reported to contain hydroxylated fatty acids⁷⁶, which would be consistent with its reduced TLC mobility. Overall, VT2 binds Gb₃ less effectively than VT1⁸⁴ and this was reflected in the TLC overlay both for renal and lung Gb₃. This procedure, however, is semiquantitative and not optimum for distinguishing the relative binding of Gb₃ isoforms⁸². Kinetic binding analysis and Gb₃ characterization has yet be performed to determine whether differential binding to Gb₃ lipid isoforms could provide the basis of the differential tissue recognition by VT1 and VT2.

Receptor analogue treatment

It is clear that our first attempts to prevent VT/Gb₃ binding in vivo have failed. Despite the fact that adaGb₃ inhibited VT2/Gb₃ binding in vitro, co-administration exacerbated, rather than attenuated, VT2-induced disease. This was particularly evident when less than 1 LD₅₀ VT2 was given, when all the VT2-treated mice survived, but all the VT2 + adaGb₃ mice died. This correlates with evidence of "novel" renal pathology, despite the fact that some of the VT2-alone pathologies were reduced by adaGb₃ co-administration. The additional pathologic features seen in the presence of adaGb₃ may result from acceleration of the VT2-induced pathology, rather than new lesions per se. The unusual properties of adaGb₃ include that despite its water solubility, the TLC mobility is not greatly affected and VT binding is effectively monitored by TLC overlay. Such solubility properties might result in complex effects in vivo. Our in vivo results are similar to studies attempting to use lyso-Gb₃ to protect mice from VT2, which found symptoms were increased rather than alleviated by this treatment¹⁰⁰.

Tissue Gb₃ is heterogeneous within its lipid moiety and membrane environment and therefore may bind VTs differentially⁸¹. We would interpret our results in terms of inhibition of lower affinity VT2/Gb₃ interactions (and hence increasing the serum half-life as observed) to allow more VT2 to bind to high-affinity (renal) receptors, thus increasing/altering pathology. The organization of Gb₃ at the plasma membrane into lipid microdomains has recently been found to be important in determining VT1 sensitivity¹⁰¹. Such organization may affect the relative Gb₃-binding affinity of VT1 (and VT2).

At specific Gb₃ concentrations, evidence for cooperative VT1-receptor binding has been found¹⁰². If such binding also occurs for VT2, an alternative explanation for the effect of adaGb₃ might be that binding of adaGb₃ to a proportion of the Gb₃ binding sites within the B subunit pentamer cooperatively increases the Gb₃ binding affinity of the residual sites to increase VT2 pathogenesis. However, this explanation would not be consistent with the increased serum half-life seen for VT2 in the presence of adaGb₃.

While these results are disappointing, they indicate that adamantyl glycolipids are active in an in vivo system to reveal further complexity of VT2-receptor targeting. These studies must also serve as a caution in the development of receptor analogues for clinical use.

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Acknowledgments

This study was supported by CIHR grant # MT13073 (to C.A.L) and by NIH/MBRS grant # GM08241 and NIH/RIMI grant # RR11598 (M.D.M). The authors are indebted to Dr. Ernest Cutz, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, for evaluation of the lung histopathology. We thank Ms. Lily Morikawa and Mr. Davin Chark from the Hospital for Sick Children for preparing and immunostaining some histological sections and Ms. Bonnie Welsh for assistance in animal procedures.