Induction of interleukin-1ß, tumour necrosis factor-{alpha} and apoptosis in mouse organs by amphotericin B is neutralized by conjugation with arabinogalactan

Rama Falk1, Moshe Hacham1, Abraham Nyska2, Julie F. Foley2, Abraham J. Domb3 and Itzhack Polacheck1,*

1 Department of Clinical Microbiology and Infectious Diseases, The Hebrew University—Hadassah Medical Center, PO Box 12000, Jerusalem 91120, Israel; 2 Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709, USA; 3 Department of Medicinal Chemistry and Natural Products, The Hebrew University of Jerusalem—School of Pharmacy, PO Box 12065, Jerusalem, Israel


* Corresponding author. Tel: +972-2-677-6592; Fax: +972-2-641-9545; Email: itzhack.polacheck{at}huji.ac.il

Received 11 December 2004; returned 18 January 2005; revised 1 February 2005; accepted 7 February 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To investigate the possibilities that: (i) organ toxicity of amphotericin B-deoxycholate (AMB-DOC) is related to induction of interleukin-1ß (IL-1ß), tumour necrosis factor-{alpha} (TNF-{alpha}) and apoptosis in target organs; and (ii) the reduced toxicity resulting from the conjugation of AMB with water-soluble arabinogalactan (AMB-AG), is related to modulation of these parameters.

Methods: Organ expression of IL-1ß and TNF-{alpha} was evaluated by enzyme-linked immunosorbent assay (ELISA) in mouse organ biological fluids and in situ by immunohistochemistry. Tissue damage was evaluated histologically, and apoptosis was demonstrated by terminal dUTP nick end-labelling (TUNEL) staining. AMB-AG conjugate was compared with the micellar (AMB-DOC) and liposomal (AmBisome) AMB formulations.

Results: Treatment with AMB-AG or AmBisome caused no observable histopathological damage in the kidneys. In contrast, treatment with AMB-DOC resulted in disruptive changes and apoptosis in renal tubular cells. These effects were found to correlate with induction of high levels of IL-1ß and TNF-{alpha} in kidney lysates. Unlike AMB-AG, AMB-DOC also induced enhanced IL-1ß and TNF-{alpha} expression in lysates of lungs, brain, liver and spleen. The marked elevation of these inflammation-apoptosis-promoting cytokines after treatment with AMB-DOC may mediate its systemic and local renal damage. Treatment with AMB-AG (but not AmBisome) appears to uniquely modulate the in situ expression of IL-1ß and enhance secretion of TNF-{alpha} in kidneys, effects possibly involved in prevention of apoptosis.

Conclusions: AMB-related toxicity is associated with induction of IL-1ß, TNF-{alpha} and apoptosis in organs. These effects were not observed with AMB-AG conjugate, suggesting its potential as a safer formulation for therapy.

Keywords: AMB toxicity , cytokines , AMB formulations , antifungals


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amphotericin B-deoxycholate (AMB-DOC) is frequently used for treating invasive fungal infections caused by a wide range of fungi,14 particularly in immunocompromised hosts, such as patients who have undergone organ transplantation, cancer patients and patients with AIDS.58 The use of AMB-DOC is limited by the frequent occurrence of adverse side effects including infusion-related fever, chills, hypotension, thrombophlebitis, dyspnoea and nausea.9 In addition, AMB-DOC frequently causes renal failure, and nephrotoxicity is one of the major limiting factors for the duration of AMB therapy.10,11

Many of the systemic side effects generated by AMB-DOC can be explained by induction of the proinflammatory, alarm-response cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-{alpha} (TNF-{alpha}), which are important mediators of the acute phase response and play crucial roles in initiation and potentiation of immune/inflammatory responses.1214 These cytokines are also endowed with an outstanding tissue-damaging potential, including induction of apoptosis.1214 AMB potently induces IL-1ß and TNF-{alpha} in vivo and in vitro, especially in macrophages.1517 In addition, these infusion-related effects of AMB are reduced by use of AMB lipid formulations17,18 which induce lower levels of these cytokines. Although it is not yet fully understood how AMB-DOC generates renal damage, AMB-induced apoptotic cellular damage could be a relevant mechanism.19

Several strategies, relying primarily on modifications of the delivery system, have been used to improve the therapeutic effectiveness of AMB and at the same time reduce its toxicity. As a result, three lipid formulations of AMB are now commercially available (AMB-lipid complex, Abelcet; AMB colloidal dispersion, Amphocil; and liposomal AMB, AmBisome), among them AmBisome is the most frequently used. Although this formulation is less toxic, its cost is extremely high and its overall success rate has not been fully determined.1,2

One of the techniques used for improving drug performance and reducing toxicity is conjugation to a polymeric carrier.20,21 Our previous studies revealed that conjugation of AMB to arabinogalactan (AG) generated a highly water-soluble amphotericin B–arabinogalactan (AMB-AG) conjugate, which was formulated for injection.22,23 The high water solubility of this conjugate determines distinct variations in its pharmacokinetic characteristics and tissue distribution in comparison with other AMB formulations.24 The AMB-AG conjugate, like the liposomal formulation (AmBisome), was more effective than AMB-DOC for prolonging survival in three different murine models: candidiasis, cryptococcosis23 and aspergillosis,24 and highly effective in treating leishmaniasis in a mouse model.25 The AMB-AG conjugate was significantly less toxic compared with AMB-DOC [maximal tolerated dose (MTD) 50 versus 4 mg/kg, in mice23 ]. In the present investigation, we studied the mechanism of toxicity of AMB-DOC and its blockage by conjugation of AMB to AG. We provide evidence that the differences in the potential of AMB-DOC, AmBisome and AMB-AG to injure murine kidneys, the major target organ of AMB toxicity, are reflected by the extent of drug-induced apoptotic damage and the specific patterns of associated IL-1ß and TNF-{alpha} expressions in this organ. The potential of AMB-AG and AMB-DOC for inducing different patterns of IL-1ß and TNF-{alpha} expression is also demonstrated in other organs. AMB-AG seems to be superior to AMB-DOC, as it does not induce renal apoptotic damage, possibly through modulation of organ cytokines to specific, apoptosis-inhibiting patterns of expression.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of the AMB formulations

AMB (950 U/mg; Dumex, Copenhagen, Denmark) was conjugated by reductive amination through its primary amine by a Schiff base reaction with oxidized AG aldehyde [99% pure; Larex (St Paul, MN, USA) with an average mol. wt of 20 000 (1 g; 0.0599 mol of saccharide units)] followed by reduction with sodium borohydride. The resultant AMB-AG conjugate contained 20% by weight AMB as evaluated by UV absorption at 385 and 416 nm, as previously described.22,23

The AMB-AG conjugate was dissolved in 5% dextrose and filter sterilized prior to injection through a 0.2 µm cellulose acetate non-pyrogenic sterile filter (Schleicher & Schuell, Dassel, Germany). AMB-DOC (Fungizone; Squibb, Middlesex, UK) and AmBisome (Gilead Sciences, Foster City, CA, USA) were prepared according to the manufacturers' instructions. All formulations were tested for endotoxins using the Limulus amebocyte lysate assay and checked for sterility using BACTEC apparatus, as previously described;23 they were kept at 4°C and used within 24 h.

Animal studies

To simulate clinical conditions in which AMB toxicity occurs, we analysed the expression of IL-1ß and TNF-{alpha} in mouse organs after multiple treatments with various doses of the AMB-AG conjugate and AMB-DOC.

Each of these AMB formulations (AMB-AG and AMB-DOC) was administered intravenously by single bolus injections of 0.2 mL for five consecutive days to groups of 10 male albino BALB/c immunocompetent non-infected mice weighing 20–25 g (this strain and type of mice were used in all experiments). The dose was 1 mg/kg per day in the AMB-DOC micellar formulation (due to the high toxicity of higher doses of AMB-DOC), and 1 or 5 mg/kg per day for the AMB-AG conjugate. On the sixth day after the first injection was started, the mice were sacrificed by CO2 asphyxiation, and the brain, lungs, liver, heart, kidneys and spleen were removed, weighed and handled as described below. Two groups of control mice were included: the first group received 5% dextrose and the second group received 20 mg/kg per day AG (representing a dose of 5 mg/kg per day of AMB-AG conjugate), according to the above mentioned experimental protocol. Each experiment was conducted in triplicate. The mice were kept in a specific-pathogen-free environment in the animal house of the Hebrew University—Hadassah Medical School. In additional experiments, the mouse kidneys were excised 4 h after a single intravenous (iv) injection of 4 mg/kg of each of the three AMB formulations—AMB-AG, AMB-DOC and AmBisome.

All procedures, care and treatment of mice were in accordance with the principles of humane treatment outlined by the Guide for the Care and Use of Laboratory Animals of the Hebrew University, and were approved by the Committee for Ethical Conduct in the Care and Use of Laboratory Animals.

Preparation of organ biological fluids

For the preparation of lysates, organs were excised and removed on ice under aseptic conditions, rinsed three times in cold PBS, weighed and subsequently rinsed three times in a large volume of cold, antibiotic-containing (100 U/mL of penicillin and 100 µg/mL of streptomycin) serum-free RPMI-1640 (Sigma Chemical Co., St Louis, MO, USA). Organs were cut into small pieces (~3 mm3) and subsequently homogenized on ice (0.1 g of tissue/mL in complete RPMI medium), using a Heidolph RZR-1 homogenizer (Germany). The collected homogenate was cleared of debris by centrifugation (1800 g, 10 min) and lysates were sterilized by filtration, using polysulphone 0.45 µm pore-size, non-pyrogenic sterile filters (Schleicher & Schuell), checked for sterility and endotoxins (as described above for the AMB-AG preparations), aliquotted, and stored at –20°C. To minimize potential non-physiological effects caused by released tissue proteases, lysates were prepared from freshly harvested organs/tissues without additional incubation in cell-culture medium. A mixture of protease/oxygenation inhibitors [0.2 mM phenylmethyl-sulphonyl fluoride (PMSF), 10 µg/mL of aprotinin, 10 µg/mL of leupeptin, 10 µg/mL of pepstatin and 0.5 mM dithiothreitol (DTT), all purchased from Sigma] was added during the preparation of organ samples to neutralize the effects of tissue proteases.

For the preparation of kidney conditioned medium, kidneys were removed on ice under aseptic conditions, weighed and rinsed as described above. The whole organs were incubated for 24 h in serum-free, antibiotic-containing RPMI medium (0.1 g tissue/1 mL of medium) at 37°C in the presence of the protease-inhibiting factors, as mentioned above. Supernatants were then harvested, centrifuged (1800 g for 10 min), sterilized and tested for endotoxins and sterility as described for lysate preparation. The supernatants were stored at –20°C until assayed. Standardization was achieved by diluting the biological fluids obtained from the different organs in proportion to tissue weight.

Protein level measurements in organ biological fluids provided an additional criterion for standardization of the different samples. This was performed using the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany). A mean value of 245 µg/mL, with negligible variation, was found in lysates from the different organs. A value of 90 µg/mL was found in kidney conditioned medium.

Analyses of organ lysates aimed to detect cytokine expression in the intracellular/membrane-associated compartments under steady-state conditions. On the other hand, analyses of kidney conditioned medium aimed to detect cytokines secreted by stressed tissue, as entailed by the relative hypoxia and nutrient-deficiency, characterizing the ex vivo organ culturing conditions. Assessment of cytokine expression under such conditions might better reflect the tissue response under the prolonged stress inflicted by the fungal infection and AMB treatment on the kidney. Application of the protease inhibitors aimed to minimize the danger of tissue disruption and cytokine degradation during the period of ex vivo organ culturing; indeed, after 24 h of incubation, no gross tissue histological alterations were noticed in the cultured kidneys (data not shown). In any case, our main purpose in this analysis was to highlight possible relative differences between the various AMB formulations regarding their distinct induction of organ cytokines (rather than point to specific absolute tissue concentrations).

Analysis of IL-1ß and TNF-{alpha}

The protein levels of IL-1ß and TNF-{alpha} in organ-derived biological fluids were determined by the enzyme-linked immunosorbent assay (ELISA).26 ELISA reagents for IL-1ß were purchased as a DuoSet ELISA development system from R&D Systems (Minneapolis, MN, USA) and for TNF-{alpha} were purchased as an Opt EIA cytokine ELISA set from BD Sciences (San Diego, CA, USA). The lower limits of detection were 15.6 and 7.8 pg/mL, respectively.

Immunohistochemistry—demonstration of IL-1ß

Organs were excised 4 h after a single treatment with the various AMB formulations (4 mg/kg), fixed in formalin, and embedded in paraffin. For immunohistochemical staining, organ sections (4 µm thick) were serially deparaffinized with xylene and rehydrated with ethanol. After washing in PBS, the sections were incubated for 1 h in Cas-Blocker solution (0.5% w/v casein powder and 0.1% w/v sodium azide in PBS, Sigma; adjusted to pH 7.4 and stored at 4°C). All incubations were performed overnight at 4°C in humidified chambers. Goat polyclonal antibody against mouse IL-1ß (R&D Systems, Inc., Minneapolis, MN, USA) that served as the primary detecting antibody was diluted in CAS-Blocker to 5 µg/mL. Following extensive washing with 0.2% Triton X-100 in PBS (three consecutive 15 min immersions), goat anti-mouse biotinylated IgG (secondary antibody; Zymed Laboratories, San Francisco, CA, USA) diluted 1:200 in Cas-Blocker solution was applied for 30 min. After three additional washings in the same solution, the endogenous peroxidase activity of sections was quenched with 9:1 methanol/3% H2O2 solution (slides immersed for 15 min). The slides were washed in Triton X-100/PBS solution and reacted with avidin-biotinylated horseradish peroxidase complex (ABC; Zymed) for 30 min. Subsequently, 3,3'-diamino-benzidine (DAB; Sigma) was added, yielding a specific reddish-brown staining. After stopping the dye reaction by washing in distilled water, the sections were counterstained with haematoxylin and eosin (H&E) and mounted in Eukitt Mounting Medium (O. Kindler Gmb H&Co, Freiburg, Germany). IL-1ß demonstration in mononuclear cells in the spleen and liver of lipopolysaccharide (LPS; Sigma)-injected mice (known to express IL-1ß),27 served as a positive control (iv injection of 100 µg of LPS per mouse and organ excision after 4 h). Specific staining for IL-1ß was not demonstrated in these organs in unstimulated mice, thus serving as a negative control (also negative by ELISA). Specificity of IL-1ß staining was verified by omission of the primary (capture) antibody and its treatment with recombinant IL-1ß prior to antibody use in the test (resulting in either abrogation or marked reduction, respectively, of staining; data not shown).

Histopathological evaluation

Mice were treated with 4 mg/kg of each of the three AMB formulations by intravenous single bolus injection of 0.2 mL (10 mice per group). The kidneys were excised 4 h post drug administration, fixed in 10% neutral-buffered formalin, processed and embedded in paraffin. Sections 5–6 µm thick were stained with H&E for microscopic examination, conducted by a board-certified toxicological pathologist. Each mouse kidney section was evaluated by an investigator blinded to the identity of the treatment group, and scored for histopathological changes according to the best practices guideline for toxicological histopathology.28 Based upon the percentage of damaged renal parenchyma (tubular cells), severity of the lesions was graded as: minimal, up to 25%; mild, 26–50%; moderate, 51–75%; or marked, > 76% of involvement. The experiments were performed in triplicate; 10–20 slides were examined for each treatment.

Staining for apoptosis

Mice (10 per group) were treated with a single dose (4 mg/kg) of each of the three AMB formulations, as described above. The kidneys were excised after 4 h of treatment, fixed in Bouin solution for 24 h and then washed daily with 70% ethanol for three consecutive days. Apoptosis was determined using the ApopTag In Situ Apoptosis Detection Kit (Intergen Company, Purchase, NY, USA), which was used to detect apoptotic cells by the terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling (TUNEL) technique.29 Apoptotic cells were identified by a brown to black nuclear pigment. For estimation of the level of apoptosis, the TUNEL-positive renal tubular epithelial cells, as well as unstained cells, were scored in 10 different fields at x400 magnification. The apoptotic index was expressed as the percentage of total cells scored. One negative control slide was prepared from every animal examined for TUNEL labelling.

Statistical analysis of results

The experiments shown are a summary of the data from at least three experiments (pool of 30 mice) and are presented as means ± SD. Statistical evaluation of the results was performed using the unpaired, two-tailed Student's t-test for single comparisons or ANOVA for multiple comparisons. The results were considered significant when P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IL-1ß and TNF-{alpha} expression in mouse organs after treatment with different AMB formulations

The expression of IL-1ß and TNF-{alpha} was studied by ELISA in various organ-derived biological fluids, and was further characterized by immunohistochemistry as described in the Materials and methods section.

ELISA analyses. Repeated AMB-DOC and AMB-AG injections of mice led to detection of overall significant differences in the expression of IL-1ß and TNF-{alpha} in lysates obtained from the brain, kidneys, liver, spleen, heart and lungs. Figure 1(a) shows that administration of 1 mg/kg per day AMB-DOC induced significantly higher amounts of IL-1ß, compared with equidose treatment with the AMB-AG conjugate in the kidneys, spleen and liver (P < 0.01). A similar trend was evident in the brain and lungs, though not statistically significant. Even repeated treatment for five consecutive days with the higher dose of AMB-AG (5 mg/kg per day) did not significantly raise the IL-1ß level above the control (with the exception of the heart) and thus was significantly lower than that obtained with the low dose of AMB-DOC (1 mg/kg per day; P < 0.01).



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Figure 1. Induction of IL-1ß (a) and TNF-{alpha} (b) as measured by ELISA in various organ lysates of mice treated with five consecutive daily iv doses of AMB-DOC (1 mg/kg per day, white bars), AMB-AG conjugate (5 mg/kg per day, striped bars; 1 mg/kg per day, black bars), Control-D (5% dextrose, dotted bars) and Control-AG (20 mg/kg per day AG, grey bars). Organs were excised 6 days post-injection.

 
The same trend was observed when TNF-{alpha} expression was assessed. AMB-DOC (1 mg/kg per day) induced significantly higher TNF-{alpha} levels than each dose of the AMB-AG conjugate (1 and 5 mg/kg per day) in the brain, kidneys, liver and lungs (P < 0.01) (Figure 1b). No significant difference was found between the levels of TNF-{alpha} induced by AMB-DOC and the two doses of AMB-AG in the heart and spleen (Figure 1b).

The level of both cytokines after stimulation with AG alone was very similar to that obtained after stimulation with 5% dextrose (Figures 1 and 2).



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Figure 2. Induction of IL-1ß (a) and TNF-{alpha} (b) as measured by ELISA, in kidneys of mice intravenously injected with 4 mg/kg of AMB-DOC, AMB-AG conjugate and AmBisome. Control-D, 5% dextrose; Control-AG, 20 mg/kg AG. ‘LYS’ (black bars) stands for organ lysates, ‘CM’ (grey bars) stands for organ conditioned medium. The kidneys were excised 4 h post-infection.

 
The pattern of cytokine expression was further studied in the kidneys, the main target organ of AMB toxicity, including analyses of the kidney conditioned medium (in addition to the lysate). In the kidney lysates, the level of IL-1ß induced by a single dose of AMB-DOC (4 mg/kg) was significantly higher (P < 0.01) than that observed after administration of equidoses of both the AMB-AG conjugate and AmBisome (Figure 2a); the level of IL-1ß detected after treatment with the AMB-AG conjugate was comparable with the control level (Figure 2a). The level of TNF-{alpha} induced by AMB-DOC in the kidney lysates was higher than that demonstrated for AmBisome and AMB-AG conjugate treatment (Figure 2b, P < 0.05). However, in the kidney-conditioned medium, this difference was not noticed; here, the AMB-AG conjugate induced the relative highest TNF-{alpha} level (Figure 2b, P < 0.01) compared with the other AMB formulations. In contrast, the levels of IL-1ß in kidney-conditioned medium after treatment with the different AMB formulations were comparable to controls (Figure 2a).

Immunohistochemistry. IL-1ß expression was markedly demonstrated in the kidney tubular cells of mice treated with AMB-AG conjugate [1, 2, 4 (Figure 3) and 10 mg/kg] and a dose–response effect was observed (data not shown). The pattern of IL-1ß staining was diffuse and included the outer medulla–inner cortex region, the site of maximal AMB-DOC-induced apoptotic damage (Figure 3, the original reddish-brown staining specific for IL-1ß is shown in dark-black). The level of AMB-DOC-induced IL-1ß in the kidney was relatively much more reduced (data not shown), whereas AmBisome did not induce IL-1ß at any of the doses used (data not shown). The pattern of the in situ expression of IL-1ß in the kidney, induced by AMB-AG, was similar to that obtained in kidneys of the BALB/c mice, used in this work, after LPS stimulation (staining performed in parallel to the spleen and liver, data not shown). In addition, a similar IL-1ß-specific staining was shown in kidneys of LPS-stimulated C57BL/6 mice (M. Hacham, S. Argov & R. N. Apte, unpublished results). No TNF-{alpha} expression could be detected in the outer medulla–inner cortex region (the site of maximal AMB-DOC-induced tubular cell apoptosis, data not shown). Some of our data indicate that TNF-{alpha} may be expressed in the middle region of the cortex; this possible expression should be further investigated using additional, more stringent controls.



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Figure 3. Immunohistochemical staining of mouse kidneys for IL-1ß demonstrating positively-stained tubular cells in mice treated with 4 mg/kg AMB-AG conjugate (a), compared with untreated mice in which no staining was demonstrated (b). The specific staining for IL-1ß, originally demonstrated by a reddish-brown hue, is presented here in dark-black. Kidneys were excised 4 h post-injection. Magnification, x400.

 
Histopathological evaluation of kidneys of mice treated with AMB formulations

Histopathological damage was detected in kidneys of mice treated with 4 mg/kg AMB-DOC. This consisted of epithelial cell necrosis of varying degrees located in the inner cortex and outer medulla (up to 75% of the medulla area and 25% of the cortex area; only the extreme changes are shown in Figure 4a). The necrotic cells displayed a homogenous eosinophilic cytoplasm associated with pyknosis or complete loss of the nuclei (Figure 4a). Proteinaceous debris was present in the tubular lumen. No damage was observed in the kidneys of other groups of mice treated with the other AMB formulations (AMB-AG, Figure 4b; AmBisome, data not shown).



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Figure 4. H&E staining of mouse kidney sections after treatment with different AMB formulations. Histological findings are shown in black and white, and represent the original H&E staining. The mice were iv injected with 4 mg/kg of (a) AMB-DOC and (b) AMB-AG conjugate and the kidneys were excised 4 h post-injection. In (a) arrows point to necrotic tubular epithelium. No damage to the renal epithelium was noted in the AMB-AG-treated mice (b). Magnification, x250.

 
Apoptosis in mouse kidneys treated with various AMB formulations

As shown in Figure 5, single treatment with the AMB-AG conjugate caused no significant apoptosis (apoptotic index < 0.2%) with all dosages used (Figure 5b shows a typical histological section, in which no apoptotic cells were found). Similarly, treatment with AmBisome did not generate significant apoptosis in the kidney (data not shown). In contrast, a considerable level of tubular cell apoptosis was observed in mice treated with 4 mg/kg AMB-DOC (Figure 5a, apoptotic index=3.67). In Figure 5(a), the TUNEL-stained nuclei shown in dark-black represent the original brown-black staining. The apoptotic cells were distributed in the region of the junction of the outer medulla and inner cortex (Figure 5a). The induction of apoptosis was dose-dependent and was not observed after treatment with lower doses of AMB-DOC. The highest dose tested for the AMB-AG conjugate (10 mg/kg) and a reduced dose of AmBisome (5 mg/kg) showed similar very low apoptotic indices (0.19 and 0.17, respectively) that were 20-fold lower than those obtained with 4 mg/kg of AMB-DOC (3.67, P < 0.01).



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Figure 5. Apoptotic cells detected by the TUNEL method in mouse kidney sections. The mice were iv injected with 4 mg/kg of AMB-DOC (a) and AMB-AG conjugate (b), and the kidneys were excised 4 h post-injection. No apoptotic cells are demonstrated after treatment with AMB-AG in panel (b), contrasted by the numerous TUNEL-stained apoptotic nuclei found after treatment with AMB-DOC (a). Arrows point to stained nucleus in apoptotic cells. The dark-black colour represents the original brown-black staining of nuclei in apoptotic cells. Magnification, x400.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the availability of new azoles and caspofungin,30 AMB remains a mainstay in treatment of invasive fungal infections mainly due to its wide spectrum fungicidal activity and low resistance development.18 However, the use of AMB-DOC is considerably hampered by the adverse side effects it causes, chiefly nephrotoxicity.10,11 Since the mechanisms underlying AMB-DOC nephrotoxicity are poorly understood,10,11 their elucidation and possible modulation could improve our capability to treat invasive fungal infections. We recently described a novel injectable AMB-AG conjugate that was much safer and more therapeutically effective in murine models of various fungal systemic infections than the conventional micellar AMB-DOC.2225 In this study, we provide evidence that indicates that AMB-DOC has the potential to induce apoptosis in the kidney, in association with a generalized up-regulation of IL-1ß and TNF-{alpha}. We also demonstrate that conjugation of AMB to the AG polysaccharide prevents these effects.

After repeated treatment with AMB-DOC, we observed a prominent elevation of the intracellularly-expressed IL-1ß and TNF-{alpha} in organ lysates (Figure 1). In the kidneys, a single treatment with one dose of AMB-DOC (4 mg/kg per day) sufficed to considerably elevate the expression of these cytokines (Figure 2) and generate apoptosis in tubular cells (Figure 5). These effects were not present when using the AMB-AG conjugate, (Figures 1, and 5), supporting a powerful tissue-preserving effect of the AMB-AG conjugate, possibly through modulation of IL-1ß and TNF-{alpha} expression in organs.

Treatment with AMB-DOC (and not with AMB-AG) was associated with marked apoptotic damage in renal tubular cells in the outer medulla–cortex border (Figure 5a). These findings highlight apoptosis as a possible mediating mechanism in AMB-DOC-induced nephrotoxicity and are in accord with reports linking AMB-DOC nephrotoxicity to apoptosis.19,31

The AMB-DOC inductions of TNF-{alpha} and IL-1ß in the kidneys suggest that these tissue-damaging and apoptosis-inducing cytokines may mediate the drug nephrotoxic effect; generation of apoptosis in renal cells by these cytokines could be one of the potential mechanisms of nephrotoxicity. Indeed, these cytokines were shown to be expressed in the kidney during stimulation by infectious agents and their components,32,33 or under ischaemic conditions.34,35 Renal production of TNF-{alpha} and TNF-{alpha}-dependent apoptosis, have been described under ischaemic conditions35 and cisplatin-induced injury in the kidney.36,37 Apoptosis in renal tubular cells was also observed after reaction of these cells with recombinant TNF-{alpha}, IL-1ß38 and LPS38 (LPS potentially inducing these cytokines in renal tubular cells). In addition, apoptosis per se, and TNF-{alpha} or IL-1ß direct effects on renal tubular cells, disturb regulation of electrolyte transport in these cells, as typically found in AMB-afflicted kidneys.39 If TNF-{alpha} plays a role in AMB-DOC-induced renal apoptosis, it could exert this effect in its membrane-bound form (as assessed in the kidney lysates)40 or secreted-soluble form.41 Moreover, TNF-{alpha} was not found by immunohistochemistry to be expressed in the outer medulla–cortex border (the site of AMB-induced apoptosis, Figure 5a), suggesting a possible action by a soluble or secreted form of TNF-{alpha}, originating outside this region. In addition, IL-1ß is typically active in its secreted form.13 Therefore, TNF-{alpha} and IL-1ß were also analysed in the kidney-conditioned medium using a similar procedure as described for the characterization of organ secreted cytokines,42,43 including TNF-{alpha}.44

Possibly, the lower level of TNF-{alpha}, found in the kidney-conditioned medium after treatment with AMB-DOC (Figure 2; relative to AMB-AG), might mediate apoptosis through preferential binding to TNFR1 expressed in the kidney45,46 with unique high affinity. In contrast, the differently-expressed TNF-{alpha} observed in the kidney-conditioned medium after treatment with AMB-AG, may not have tissue-damaging effects. The higher levels of soluble renal TNF-{alpha} after treatment with AMB-AG, might bind with different affinity to TNFR1 and also activate TNFR2, shown to be expressed in renal tissue.46 This may result in an altered signal transduction that may not generate apoptosis. Such a possible apoptosis-inhibiting potential might also be attributed to IL-1ß, apparently expressed in the kidney in a unique manner after treatment with AMB-AG, as shown by the distinct positive immunohistochemical staining (Figure 3). IL-1ß, like TNF-{alpha}, can under specific conditions, mediate dual effects with a narrow window between beneficial and harmful effects.13,47 Thus, intracellularly expressed IL-1ß could paradoxically inhibit apoptosis, through intracellular interference with proapoptotic mechanisms,48 under the conditions which could be specifically present in the kidney after treatment with AMB-AG but not with AMB-DOC.

Treatment with AmBisome, as with AMB-AG, did not result in observable kidney damage in the experiments described herein. This could suggest a similar action of these two AMB formulations in the clinical setting. However, unlike AMB-AG, AmBisome did not induce a relative increase in secretion of TNF-{alpha} or intracellular expression of IL-1ß in the kidney. As these patterns of cytokine modulation might imply the induction of active, apoptosis-negating mechanisms, it is possible that the AMB-AG-organ-sparing effect could be of a more sustained nature.

How could conjugation to a polysaccharide macromolecule, arabinogalactan, alter so markedly the potential of AMB to induce apoptosis in renal tubular cells and its associated cytokine expression? A possible mechanism could involve interference by the arabinogalactan molecule with the putative internalization of AMB into renal tubular cells and the consequent induced cytokine-mediated cellular damage. Though not yet directly shown in renal cells, AMB linked to low density lipoproteins (LDL) can be internalized into cells through LDL receptors, as shown in the non-phagocytic ovary CHO cell line,49 where it caused cell damage. Additionally, AMB concentration and the resulting toxicity increased in the kidney when the serum LDL level was elevated,50,51 similar to TNF-{alpha} expression in monocytes engulfing AMB-lipoprotein particles.50,52 Thus, conjugation of AMB to AG might obstruct both the access of the AMB molecule to the cell membrane and its binding to the LDL particles, ultimately preventing or reducing cell damage. This hypothesis is supported by the observation that after stimulation with AG alone, the levels of renal cytokines did not differ significantly from the basal levels (Figures 1 and 2), suggesting that AG does not exert an independent effect on renal cells.

This suggested AG interference with AMB binding to specific cellular receptors may also be applicable in other organs, in which neutralization of AMB-mediated induction of IL-1ß or/and TNF-{alpha} by AG conjugation was demonstrated. Reduced stimulation of tissue macrophages and/or phagocytic cells by AMB-AG in this respect, especially in the spleen, liver and lungs (organs rich in lymphoid cells), could plausibly explain the observed neutralization of AMB-induced proinflammatory cytokines. Indeed, AMB-induction of IL-1ß and TNF-{alpha} is mostly described in macrophages.1517 The putative AG binding to macrophage Toll-like receptors could be one such inhibitory mechanism, given the capability of these receptors to bind both AMB16 and mycobacterial cell wall proteoglycans, apparently, as well as AG.53 AG (as present in the AMB-AG molecule) might also interfere with additional cellular sites that serve as targets for AMB binding and generation of toxicity (including through induction of tissue-damaging proinflammatory cytokines). These sites could be present in endothelial5456 and parenchymal cells (such as hepatocytes),57 both of which have been shown to be stimulated/damaged by AMB.

In summary, in this study, we forward evidence that linkage of the polysaccharide arabinogalactan to AMB significantly decreases its renal toxicity by preventing apoptosis, possibly through modulation of the AMB-induced proinflammatory and apoptosis-promoting cytokines, TNF-{alpha} and IL-1ß. In addition, these findings shed light on the mode of action and mechanisms of AMB toxicity, and eventually could facilitate the design of new and less toxic AMB formulations, or novel strategies, aiming to minimize AMB-induced noxious tissue effects.


    Acknowledgements
 
We thank Michael Zeira for his technical assistance in performing the ELISA assays and Eliezer Rosenman and Ruth Reshef from Shaare Zedek Medical Center for their help in carrying out the immunohistochemistry staining. This work was supported in part by a grant from the Ministry of Science and Culture. A. J. D. is affiliated with the David R. Bloom Center for Pharmacy and The Alex Grass Center for Drug Design and Synthesis at The Hebrew University.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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