Accumulating Mb (myoglobin) in the kidney following severe burns promotes oxidative damage and inflammation, which leads to acute renal failure. The potential for haem–iron to induce oxidative damage has prompted testing of iron chelators [e.g. DFOB (desferrioxamine B)] as renal protective agents. We compared the ability of DFOB and a DFOB-derivative {DFOB-AdAOH [DFOB-N-(3-hydroxyadamant-1-yl)carboxamide]} to protect renal epithelial cells from Mb insult. Loading kidney-tubule epithelial cells with dihydrorhodamine-123 before exposure to 100 μM Mb increased rhodamine-123 fluorescence relative to controls (absence of Mb), indicating increased oxidative stress. Extracellular Mb elicited a reorganization of the transferrin receptor as assessed by monitoring labelled transferrin uptake with flow cytometry and inverted fluorescence microscopy. Mb stimulated HO-1 (haem oxygenase-1), TNFα (tumour necrosis factor α), and both ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) gene expression and inhibited epithelial monolayer permeability. Pre-treatment with DFOB or DFOB-AdAOH decreased Mb-mediated rhodamine-123 fluorescence, HO-1, ICAM and TNFα gene expression and restored monolayer permeability. MCP-1 (monocyte chemotactic protein 1) secretion increased in cells exposed to Mb-insult and this was abrogated by DFOB or DFOB-AdAOH. Cells treated with DFOB or DFOB-AdAOH alone showed no change in permeability, MCP-1 secretion or HO-1, TNFα, ICAM or VCAM gene expression. Similarly to DFOB, incubation of DFOB-AdAOH with Mb plus H2O2 yielded nitroxide radicals as detected by EPR spectroscopy, indicating a potential antioxidant activity in addition to metal chelation; Fe(III)-loaded DFOB-AdAOH showed no nitroxide radical formation. Overall, the chelators inhibited Mb-induced oxidative stress and inflammation and improved epithelial cell function. DFOB-AdAOH showed similar activity to DFOB, indicating that this novel low-toxicity chelator may protect the kidney after severe burns.

INTRODUCTION

In the event of skeletal muscle breakdown, which occurs subsequent to severe burns, the affected muscle mass can undergo a process termed RM (rhabdomyolysis) [1]. This leads to the release of toxic factors including skeletal Mb (myoglobin) into the circulation, where the protein is rapidly cleared and accumulates in the kidney (termed myoglobinuria) [2]. Myoglobinuria has been linked to ARF (acute renal failure) [3], and research in this field has demonstrated that accumulating Mb promotes both oxidative damage [4] and inflammation [5] within the kidney. Although the proportion of burns patients developing ARF is relatively low, mortality rates for these patients consistently remain above 80% [6]. Therefore development of therapeutic strategies to limit the extent of ARF following severe burns would be advantageous.

The molecular mechanisms that underlie Mb toxicity have been studied previously and include renal vasoconstriction, intraluminal cast formation and haem-induced cytotoxicity [7]. It is not clear whether renal tubular cast formation is causally related or rather a consequence of the pathology associated with ARF [8]. In addition, degradation of the accumulating Mb probably results in the release of free haem and its catabolism by HOs (haem oxygenases) [9] coupled with release of iron and carbon monoxide [10] and the concomitant formation of the antioxidant bilirubin [11]. Accumulating ferrous iron (Fe2+) can generate hydroxyl radicals via the Fenton reaction, which can then damage cellular targets, including lipids, protein and DNA, and contribute to enhanced oxidative stress [12].

Several studies have also linked oxidative stress and inflammatory responses in renal tissues and cells following RM [13,14]. The potential for iron to be involved in these responses has prompted research aimed at testing whether iron chelators such as DFOB (desferrioxamine B) can provide protection for renal tissues. For example, DFOB provides renal protection in an animal model of RM [15,16], indicating that the supplemented chelator preserves kidney function in the presence of extracellular Mb. Studies in cell models also implicate DFOB in preserving mitochondrial function in renal cells [17], although this may not be due to iron chelation alone, since DFOB also inhibits Mb pro-oxidative activity [18]. However, the corresponding therapeutic drug Desferal® is limited by its hydrophilic nature and short plasma half-life [19], low oral activity [20], an inability to cross cell membranes and modest nephrotoxicity [21]. Other orally available chelating agents are not as effective as DFOB in the primary goal of sequestering iron and show adverse side effects or increased nephrotoxicity [22,23].

Recently, a number of lipophilic low toxicity iron-chelating compounds were synthesized by conjugating DFOB to adamantyl derivatives [24]. Characteristically, these compounds showed similar iron-chelating properties to DFOB, but with decreased cytotoxicity towards cultured renal epithelial cells. Notably, in terms of toxicity, the conjugates of DFOB exhibit 6–15-fold greater IC50 values than DFOB under the same culture conditions [24]. The conjugates also exhibited subtle differences in their ability to efflux iron from cells, suggesting that structural elements and/or physical properties modulate their uptake and secretion from different cell types. It is envisaged that this novel class of iron chelator may potentially overcome the selected therapeutic limitations of Desferal® and show improved renal tolerance, as well as still maintaining a high iron-binding efficacy.

In the present study, we compared the ability of DFOB-AdAOH [DFOB-N-(3-hydroxyadamant-1-yl)carboxamide; structure shown in Figure 1] and its parent compound DFOB to protect cultured kidney epithelial cells in an established cell model of RM that mimics urinary Mb levels detected in severe electrical-burn-induced muscle myolysis [25].

Chemical structures of the therapeutic iron chelator DFOB (1) and DFOB-AdAOH (2)

Figure 1
Chemical structures of the therapeutic iron chelator DFOB (1) and DFOB-AdAOH (2)

Arrows indicate the hydroxamic acid moiety adjacent to the terminal amine group in each compound.

Figure 1
Chemical structures of the therapeutic iron chelator DFOB (1) and DFOB-AdAOH (2)

Arrows indicate the hydroxamic acid moiety adjacent to the terminal amine group in each compound.

EXPERIMENTAL

Materials

Chemicals were of the highest quality available. DFOB mesylate (95%) and cell culture materials were from Sigma–Aldrich. The DFOB–adamantane conjugate DFOB-AdAOH was synthesized and purified by HPLC as described previously [24]. Chelators were analytically pure as defined by HPLC. Fresh stock solutions of DFOB and DFOB-AdAOH (final concentration 1 mM) were prepared in DMSO. Experiments were carried out with chelators present at a final concentration 100 μM, as this was within the IC50 toxic level for each agent [24].

Cell culture

Kidney epithelial cells [MDCK II (Madin–Darby canine kidney II); A.T.C.C.] were cultured as described previously [25]. Plates of cells were randomly allocated for control or iron chelator pre-treatment. Cells were washed twice in PBS (pH 7.4) and treated with vehicle (DMSO, control) or 100 μM iron chelator diluted in HPSS (Hepes-buffered physiological salt solution; 22 mM Hepes, pH 7.4, 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 0.16 mM H3PO4, 5 mM NaHCO3 and 5.6 mM D-glucose). After 2 h at 37°C, cells were washed and then treated with 100 μM Mb or vehicle (as a control). In rats with glycerol-induced ARF, urinary Mb can reach ~2 mM [26]. However, despite significant myolysis of skeletal muscle in cases of severe electrical burns, urinary Mb is only within the range of ~50–60 μM [25]. Therefore in the present study we chose to employ a dose of 100 μM Mb as an insult to the cultured epithelial cells to mimic the renal pathology induced by severe burns. Under these conditions, cell viability in control and Mb-treated cells remained unchanged after 24 h [25] and this time point was judged as suitable to make the biochemical and molecular comparisons described below.

Cell model of RM

Equine heart ferric Mb (Sigma) was used to establish the cell model of RM. Mb solutions were freshly prepared in PBS, sterilized by passage through 0.2 μm pore-size filters (Millipore) and then standardized using ϵ409=188 mM−1·cm−1 [27]. All solutions prepared in this way displayed a Soret band at 409 nm assigned as ferric haem with no other absorbance in the range 410–425 nm, indicative of the absence of ferrous-oxy-Mb. The stock solution of Mb was then diluted in culture medium to a final concentration of 100 μM for use in cell studies. After 24 h, cells were washed twice with PBS and harvested for the assays outlined below. Where required, cells were harvested with 0.1% trypsin and pellets were isolated by centrifugation (358 g for 4 min, 4°C).

Oxidative stress response

To determine oxidative stress during experimental RM, cells were pre-treated with iron chelator or vehicle alone and incubated with Mb for 24 h as described above. Prior to harvest (~4 h), cells were exposed to 50 μg/ml non-fluorescent DHR-123 (dihydrorhodamine-123; Invitrogen). In some experiments, Mb was removed prior to incubation with DHR-123. Oxidation of DHR-123 yields the fluorescent product R-123 (rhodamine-123). The cellular probe DHR-123 reacts non-specifically with intracellular ROS (reactive oxygen species) in a reaction mediated by peroxidase, cytochrome c or Fe2+ and thus is a useful fluorigenic probe for detecting enhanced oxidative stress in cells and tissues [28]. Mean fluorescence intensity was measured by flow cytometry (FACSCalibur; BD Biosciences) with excitation at 488 nm and emission at 540 nm. For each experiment conducted, 10000 events were recorded before assessment of the data using standard FACSCalibur software.

Gene regulation

Isolation of total mRNA and assessment of gene regulation was determined by quantitative real-time RT (reverse transcription)–PCR. Briefly, MDCK II cells were lysed and total RNA was isolated with a commercial kit according to the manufacturer's instructions (GenElute; Sigma). Equal amounts (1 μg) of RNA were primed with oligo(dT)15 primers and reverse-transcribed using a cDNA synthesis kit (Bioline). Amplification of cDNA was performed in a total volume of 15 μl of SYBR Green I Mastermix (Quantace; Bioline) containing appropriate primers using a Roche LightCycler 480. After initial denaturation (95°C for 10 min), 40 PCR cycles were performed using the following conditions: 95°C, 15 s; 60°C, 15 s; and 72°C, 15 s followed by a melt-step (55–95°C). Primer pairs used for the assessment of β-actin, HO-1, TNFα (tumour necrosis factor α), NFB (nuclear factor κB), VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) levels are shown in Table 1. Relative gene expression was assessed using the second derivative maximum method and normalized to the corresponding β-actin response for the same sample. Gene expression in the control sample was arbitrarily assigned a unitary value and gene response was expressed as a fold change relative to the control.

Table 1
Forward and reverse primer sequences used in gene analysis

Primers were obtained from Sigma and diluted to 10 μM before use. Annealing temperature was 60°C for all primer sets employed.

GeneSenseAnti-sense
β-Actin 5′-AGCCATGTACGTAGCCATCC-3′ 5′-CTCTCAGCTGTGGTGGTGAA-3′ 
HO-1 5′-GCGTCGACTTCTTCACCTTC-3′ 5′-GGTCCTCAGTGTCCTTGCTC-3′ 
TNFα 5′-GAGCCGACGTGCCAATG-3′ 5′-CAACCCATCTGACGGCACTA-3′ 
NFB 5′-AACCCGTAGTGTCAGATGCC-3′ 5′-GGACGAACACAGAGGTTGGT-3′ 
VCAM-1 5′-CAACTGAGTGGCCCCCTAG-3′ 5′-GAGATCATTGCCATTCAGCA-3′ 
ICAM-1 5′-AGAGAGGCTGCACTCCACAG-3′ 5′-GCTCACTCAGGGTCAGGTTG-3′ 
GeneSenseAnti-sense
β-Actin 5′-AGCCATGTACGTAGCCATCC-3′ 5′-CTCTCAGCTGTGGTGGTGAA-3′ 
HO-1 5′-GCGTCGACTTCTTCACCTTC-3′ 5′-GGTCCTCAGTGTCCTTGCTC-3′ 
TNFα 5′-GAGCCGACGTGCCAATG-3′ 5′-CAACCCATCTGACGGCACTA-3′ 
NFB 5′-AACCCGTAGTGTCAGATGCC-3′ 5′-GGACGAACACAGAGGTTGGT-3′ 
VCAM-1 5′-CAACTGAGTGGCCCCCTAG-3′ 5′-GAGATCATTGCCATTCAGCA-3′ 
ICAM-1 5′-AGAGAGGCTGCACTCCACAG-3′ 5′-GCTCACTCAGGGTCAGGTTG-3′ 

Endocytosis of fluorescently labelled transferrin

Preparations of MDCK II cells were supplemented with 100 μM iron chelator or vehicle (DMSO) alone and then either exposed to Mb or vehicle (PBS) as indicated in the Figure legends. After 24 h, the cells were harvested and the cell pellets that were either exposed to Mb treatment or not (control) were resuspended in 1 ml of PBS and treated with transferrin conjugated to Alexa Fluor® 488 (5 μg/ml; Invitrogen). After 15 min, the cells were analysed by flow cytometry (FACSCalibur; BD Biosciences) as described previously [29].

In separate experiments, cells were grown on to sterile coverslips, treated as described above and then washed with 0.2 M acetic acid containing 0.5 M NaCl (pH 2.8), then fixed in 4% (w/v) paraformaldehyde (pH 7.5) for 15 min at 20°C. Coverslips were then mounted with Fluorescent Mounting Medium (Dako) and examined with an inverted fluorescent microscope using identical microscope settings (Axiovert 200; Zeiss) and then saved as tagged image files for further manipulation (AxioVision v4.5; Carl Zeiss).

Evaluation of monolayer permeability

Cells were seeded (1×105 cells/ml) on to six-well, 0.4 μm pore size, transparent transwells (Greiner) and cultured to 90% confluence. Following supplementation with 100 μM iron chelator or vehicle alone, cells were exposed to 100 μM Mb or vehicle as a further control (to assess the impact of the chelators alone on monolayer permeability). After 24 h, cells were treated with 2.5 μCi of [3H]inulin/ml of complete medium (Sigma) and the amount of [3H]inulin present in the upper or lower chamber was assayed with a scintillation counter (Packard-Bell) as described previously [25]. Finally, monolayer permeability was expressed as a percentage of [3H]inulin passing through the monolayer of cells.

Expression of MCP-1 (monocyte chemotactic protein 1)

For quantitative determination of CCL2 (CC chemokine ligand 2)/MCP1, adherent cells were pre-treated with iron chelator or vehicle alone and incubated in the presence or absence of Mb as described above. After 24 h of incubation, samples of cell culture medium were taken and analysed using an AlphaLISA assay (PerkinElmer). Briefly, cell supernatants were centrifuged, diluted and incubated with anti-m/rCCL2/MCP-1 acceptor beads and the corresponding biotinylated antibody anti-m/rCCL2/MCP-1 for 60 min. Then, streptavidin donor beads were added and the absorbance was measured after a 30 min incubation on an Enspire plate reader (PerkinElmer).

Oxidation of chelators DFOB and DFOB-AdAOH by peroxidases

Solutions of the individual chelators (final concentration 500 μM) were incubated with either 250 μM HRP (horseradish peroxidase, Sigma) or Mb (Sigma), dissolved in phosphate buffer (50 mM phosphate, pH 7.4) and oxidation was initiated by the addition of 500 μM H2O2 or buffer (as a negative control). Mixing the chelator with 500 μM H2O2 alone served as a further control. After a 5 min incubation at 20°C, a sample of the reaction mixture was taken and transferred into a quartz flat cell (Wilmad) for measurement with a Bruker EMX EPR spectrometer at 293 K; with parameters of: power 100 mW, microwave frequency 9.8 GHz, modulation amplitude 1 mT and sweep 84 s. EPR spectra were recorded as an average of four cumulative scans. Hyperfine couplings were obtained by spectral simulation using the simplex algorithm [30] provided in the WINSIM program (http://www.niehs.nih.gov/research/resources/software/tools/index.cfm). All hyperfine couplings are expressed in units of mT. Simulations were considered acceptable if they produced correlation factors of R=0.98. The detection limit of the stable nitroxide (TEMPO) radical was ~50 nM. The remaining sample was frozen for analysis by reversed-phase HPLC with DFOB and DFOB-AdAOH eluting at ~10 and 13 min respectively, as described previously [24]. Where required, Fe(III) was added to DFOB-AdAOH at a 1:1 molar ratio and the Fe(III)–DFOB-AdAOH complex was purified by HPLC as described previously [24]. The purified Fe(III) complex was verified by ESI-MS yielding a molecular mass of 792.5 Da (spray voltage 4.5kV, capillary voltage 35V, carrier N2 gas) and was used in further EPR studies to assess the impact of iron loading on nitroxide radical formation.

Statistical analyses

Statistical analyses were performed with Prism (GraphPad). Results are presented as means±S.D. of replicate analyses from at least three independent experiments (or as indicated in the Figure legends). Differences between data sets were assessed with one-way ANOVA using Bonferroni's multiple comparisons post-hoc test. Significance was accepted at the 95% level; P<0.05.

RESULTS

Oxidative stress in MDCK II cells exposed to Mb

Treating cultured MDCK II cells with 100 μM Mb for 24 h resulted in a 2.3-fold increase in R-123 fluorescence relative to the controls (Figure 2), whereas removal of Mb prior to addition of DHR-123 completely diminished R-123 fluorescence, indicating that added Mb promoted DHR-123 oxidation (results not shown). Pre-treatment of cells with DFOB-AdAOH significantly decreased the mean fluorescence intensity, although this did not reach baseline levels in the control. Similarly, pre-treatment of cells with DFOB before addition of Mb diminished the mean fluorescence intensity; however, this did not reach statistical significance.

Mb-stimulated oxidative stress in MDCK II cells

Figure 2
Mb-stimulated oxidative stress in MDCK II cells

Cells were treated in the absence or presence of 100 μM iron chelator and subsequently exposed to extracellular Mb for 24 h. Prior to harvest, cells were loaded with 50 μg/ml DHR-123 and the extent of R-123 fluorescence was monitored. The results represent the means±S.D. from three independent studies each performed in triplicate. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.05.

Figure 2
Mb-stimulated oxidative stress in MDCK II cells

Cells were treated in the absence or presence of 100 μM iron chelator and subsequently exposed to extracellular Mb for 24 h. Prior to harvest, cells were loaded with 50 μg/ml DHR-123 and the extent of R-123 fluorescence was monitored. The results represent the means±S.D. from three independent studies each performed in triplicate. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.05.

Gene regulation in MDCK II cells treated with Mb

The gene response for the antioxidant response element HO-1, and the inflammatory markers TNFα, NFB, ICAM-1 and VCAM-1, were monitored by quantitative real-time RT–PCR and are shown in Table 2. Consistent with extracellular Mb eliciting a heightened oxidative stress 24 h after insult (Figure 2), HO-1 expression increased significantly in response to the same Mb insult, with expression reaching 3-fold higher levels than in the control. Addition of the iron chelators DFOB and DFOB-AdAOH muted HO-1 gene expression, suggesting that the chelators inhibited the cellular oxidative response that promotes HO-1 expression [9]. Notably, no change in HO-1 gene expression was evident in cells pre-treated with the iron chelators in the absence of Mb insult.

Table 2
Gene expression in cultured kidney epithelial cells

Confluent MDCK II cells were pre-incubated with the iron chelator indicated (final chelator concentration 100 μM) or vehicle (control) in the absence or presence of 100 μM Mb. After 24 h, cells were harvested, mRNA isolated and the corresponding cDNA was probed for gene regulation as described in the Experimental section. Gene expression levels in the control were arbitrarily assigned a value of 1 and the other results were expressed relative to this level in the controls.The results represent the means (± S.D.) from three to six different cell preparations. *Significantly different to the control; P<0.05. †Significantly different to the corresponding Mb-treated group; P<0.05.

GeneControlMbMb+DFOBMb+DFOB-AdAOHDFOBDFOB-AdAOH
HO-1 1.0 (0.0) 3.0 (0.7)* 1.5 (1.1)† 1.2 (0.6)† 1.2 (0.4) 0.9 (0.3) 
TNFα 1.0 (0.0) 5.3 (2.3)* 1.7 (0.9)† 1.8 (0.9)† 1.3 (0.9) 1.2 (0.3) 
NFB 1.0 (0.0) 1.5 (0.7) 0.9 (0.2) 0.7 (0.6) 0.4 (0.2)* 0.6 (0.3)* 
ICAM-1 1.0 (0.0) 2.6 (1.5) 1.2 (0.5) 1.1 (0.5) 1.0 (0.5) 0.9 (0.0) 
VCAM-1 1.0 (0.0) 3.3 (4.8) 7.6 (13.8) 7.6 (0.9)* 1.6 (0.9) 1.4 (0.2) 
GeneControlMbMb+DFOBMb+DFOB-AdAOHDFOBDFOB-AdAOH
HO-1 1.0 (0.0) 3.0 (0.7)* 1.5 (1.1)† 1.2 (0.6)† 1.2 (0.4) 0.9 (0.3) 
TNFα 1.0 (0.0) 5.3 (2.3)* 1.7 (0.9)† 1.8 (0.9)† 1.3 (0.9) 1.2 (0.3) 
NFB 1.0 (0.0) 1.5 (0.7) 0.9 (0.2) 0.7 (0.6) 0.4 (0.2)* 0.6 (0.3)* 
ICAM-1 1.0 (0.0) 2.6 (1.5) 1.2 (0.5) 1.1 (0.5) 1.0 (0.5) 0.9 (0.0) 
VCAM-1 1.0 (0.0) 3.3 (4.8) 7.6 (13.8) 7.6 (0.9)* 1.6 (0.9) 1.4 (0.2) 

The pro-inflammatory genes TNFα, NFB and ICAM-1 all showed a trend to increased expression in the presence of extracellular Mb, but only TNFα reached statistical significance (Table 2). Interestingly, gene expression of VCAM-1 was increased in renal epithelial cells exposed to Mb and greater increases were evident in cells pre-loaded with chelators, suggesting that the chelators DFOB and DFOB-AdAOH acted in concert with Mb to induce some pro-inflammatory responses in cultured renal epithelial cells as well as down-regulating others. Importantly, pre-treatment of the cells with DFOB or DFOB-AdAOH in the absence of extracellular Mb had no material impact on the genes monitored in the present study, with the exception of NFB, which was decreased in response to incubation with the chelators (Table 2). The latter is interpreted as depletion of the intracellular pool of iron leading to decreased (oxidative) activation of the transcription factor.

Receptor-mediated endocytosis in cultured MDCK II cells

The receptor-mediated endocytosis of fluorescently labelled transferrin in the absence or presence of added Mb and with or without DFOB or DFOB-AdAOH supplementation was quantified by flow cytometry as shown in Figure 3. The presence of two sub-populations of cells was indicative of two distinct intracellular pools of transferrin in the MDCK II cells [29]. These pools were assigned previously as a slow-cycling population of transferrin receptor complex that is mobilized to the Golgi apparatus and a pool of fast-cycling transferrin receptor complex that releases transferrin in the cytoplasm [31]. The latter mechanism leads to a rapid recycling of the receptor to the plasma membrane without translocation to the Golgi [31]. Assessment of the population scatter plots in the absence of added Mb indicated that the chelators had no material impact on the distribution of cells (results not shown).

Endocytosis of labelled transferrin in cultured MDCK II cells assessed by flow cytometry

Figure 3
Endocytosis of labelled transferrin in cultured MDCK II cells assessed by flow cytometry

Mb-treated cells were incubated with fluorescently labelled transferrin (5 μg/ml) for 15 min at 37°C prior to analysis. Data shown represent histograms of MDCK II cells in the (A) absence (control) or (B) presence of Mb (100 μM) both without chelator treatment, or samples pre-treated with 100 μM (C) DFOB or with (D) DFOB-AdAOH before addition of extracellular Mb. The arrow in (B) indicates the declining peak response that was inhibited by the presence of the chelators. (E) Endocytosis of labelled transferrin (TFN) was monitored and re-expressed as a proportion of the total cell population for each of the treatment groups. The results represent the means±S.D. from three experiments. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.001.

Figure 3
Endocytosis of labelled transferrin in cultured MDCK II cells assessed by flow cytometry

Mb-treated cells were incubated with fluorescently labelled transferrin (5 μg/ml) for 15 min at 37°C prior to analysis. Data shown represent histograms of MDCK II cells in the (A) absence (control) or (B) presence of Mb (100 μM) both without chelator treatment, or samples pre-treated with 100 μM (C) DFOB or with (D) DFOB-AdAOH before addition of extracellular Mb. The arrow in (B) indicates the declining peak response that was inhibited by the presence of the chelators. (E) Endocytosis of labelled transferrin (TFN) was monitored and re-expressed as a proportion of the total cell population for each of the treatment groups. The results represent the means±S.D. from three experiments. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.001.

Exposure of the cells to extracellular Mb induced a selective reorganization of these intracellular transferrin pools, with the left-hand peak remaining largely unaffected and the right-hand peak decreasing markedly (compare Figures 3A and 3B). By contrast, pre-treatment of the cells with DFOB or DFOB-AdAOH somewhat restored the distribution of transferrin endocytosis (Figures 3C and 3D). Quantitative analysis (accounting for both peak populations) showed a distinct Mb-dependent loss in mean fluorescence intensity that was sensitive to pre-treatment of the cells with the chelators DFOB or DFOB-AdAOH (Figure 3E).

The ability of DFOB or DFOB-AdAOH to enhance transferrin uptake in the presence of Mb was verified by monitoring the receptor-mediated binding of labelled transferrin in MDCK II cells with fluorescent microscopy (Figure 4). As anticipated, exposure of cells to Mb down-regulated the endocytosis of labelled transferrin (compare Figures 4A and 4B), whereas pre-incubation with the chelators restored transferrin receptor-mediated endocytosis as judged by the increased membrane fluorescence (compare Figures 4A, 4C and 4D). Pre-treatment with the chelators alone showed a marginal increase in transferrin endocytosis relative to the control in the absence of chelators and added Mb (compare Figures 4A, 4E and 4F), consistent with repletion of the iron stores after chelator treatment.

Inhibition of endocytosis by added Mb is reversed by pre-treatment of cells with iron chelators as judged by fluorescent microscopy

Figure 4
Inhibition of endocytosis by added Mb is reversed by pre-treatment of cells with iron chelators as judged by fluorescent microscopy

MDCK II cells were seeded (1×105 cells/ml) on to glass coverslips, sub-cultured at 37°C in a 5% CO2(g) atmosphere and then incubated with fluorescently labelled transferrin (5 μg/ml) for 15 min at 37°C prior to analysis. Representative micrographs show MDCK II cells in the (A) absence (control) or (B) presence of Mb (100 μM) both without chelator pre-treatment, or samples pre-treated with 100 μM (C) DFOB or with (D) DFOB-AdAOH before addition of extracellular Mb. The chelators (E) DFOB and (F) DFOB-AdAOH marginally enhance the uptake of labelled transferrin in the absence of added Mb. Images were taken using an inverted fluorescence microscope (63× oil objective) fitted with a high-resolution colour digital imaging camera and then transformed to tagged image files and downloaded into Microsoft PowerPoint (2007) for further manipulation.

Figure 4
Inhibition of endocytosis by added Mb is reversed by pre-treatment of cells with iron chelators as judged by fluorescent microscopy

MDCK II cells were seeded (1×105 cells/ml) on to glass coverslips, sub-cultured at 37°C in a 5% CO2(g) atmosphere and then incubated with fluorescently labelled transferrin (5 μg/ml) for 15 min at 37°C prior to analysis. Representative micrographs show MDCK II cells in the (A) absence (control) or (B) presence of Mb (100 μM) both without chelator pre-treatment, or samples pre-treated with 100 μM (C) DFOB or with (D) DFOB-AdAOH before addition of extracellular Mb. The chelators (E) DFOB and (F) DFOB-AdAOH marginally enhance the uptake of labelled transferrin in the absence of added Mb. Images were taken using an inverted fluorescence microscope (63× oil objective) fitted with a high-resolution colour digital imaging camera and then transformed to tagged image files and downloaded into Microsoft PowerPoint (2007) for further manipulation.

Evaluation of monolayer permeability

Monitoring the transport of [3H]inulin across a confluent monolayer of epithelial cells provides an indication of the bulk epithelial monolayer integrity as described previously [32]. Overall, non-specific monolayer permeability decreased significantly upon Mb challenge yielding ~15% lower permeability than the control (Figure 5). Pre-treatment of the cells with the iron chelators reversed this decrease in permissiveness and restored monolayer permeability to the control levels in the absence of extracellular Mb. Notably, pre-treatment of the cells with DFOB or DFOB-AdAOH alone had no impact on monolayer permeability in the absence of an Mb insult, consistent with the low toxicity of the chelators at the selected dose.

Monolayer permissiveness decreases in MDCK II cells after exposure to extracellular Mb and added chelators inhibit this activity

Figure 5
Monolayer permissiveness decreases in MDCK II cells after exposure to extracellular Mb and added chelators inhibit this activity

Non-specific [3H]inulin transport was monitored in the absence or presence of 100 μM iron chelator in both the presence and absence of extracellular Mb (final concentration 100 μM). Monolayer permissiveness improves in the presence of iron-chelating drugs, whereas the chelators alone have no material effect on monolayer permeability. The results represent the means±S.D. from nine experiments. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.05.

Figure 5
Monolayer permissiveness decreases in MDCK II cells after exposure to extracellular Mb and added chelators inhibit this activity

Non-specific [3H]inulin transport was monitored in the absence or presence of 100 μM iron chelator in both the presence and absence of extracellular Mb (final concentration 100 μM). Monolayer permissiveness improves in the presence of iron-chelating drugs, whereas the chelators alone have no material effect on monolayer permeability. The results represent the means±S.D. from nine experiments. *Significantly different to the control; P<0.001. #Significantly different to the corresponding Mb-treated group; P<0.05.

Expression of MCP-1

The secreted protein MCP-1 displays chemotactic activity for monocytes and basophils and is considered as a biomarker of inflammation [33]. Renal cells produce MCP-1 upon stimulation through activation of the NF-κB pathway [34]. Notably, secretion of MCP-1 from MDCK II cells significantly increased (1.3-fold) in the presence of Mb compared with cells cultured in Mb-free medium. Pre-treatment of cells for 2 h with DFOB or DFOB-AdAOH inhibited these inflammatory responses and decreased MCP-1 levels to below baseline (Figure 6). Pre-treatment of the cells with DFOB or DFOB-AdAOH in the absence of Mb insult had no effect on MCP-1 levels in cultured MDCK II cells, which was consistent with the unchanged level of TNFα gene expression recorded under identical conditions (see Table 2).

Monocyte chemoattractant protein 1 expression in MDCK II cells after exposure to extracellular Mb

Figure 6
Monocyte chemoattractant protein 1 expression in MDCK II cells after exposure to extracellular Mb

Upon exposure to Mb for 24 h, cell supernatant was collected and the AlphaLISA assay was performed to determine the MCP-1 concentration. Mb increases the expression of MCP-1, whereas pre-treatment with DFOB or DFOB-AdAOH suppressed MCP-1 expression. In the absence of extracellular Mb, the iron-chelating drugs showed no material effect on MCP-1 secreted into the medium. The results represent the means±S.D. from three experiments. *Significantly different to the control; P<0.01. #Significantly different to the corresponding Mb-treated group; P<0.001.

Figure 6
Monocyte chemoattractant protein 1 expression in MDCK II cells after exposure to extracellular Mb

Upon exposure to Mb for 24 h, cell supernatant was collected and the AlphaLISA assay was performed to determine the MCP-1 concentration. Mb increases the expression of MCP-1, whereas pre-treatment with DFOB or DFOB-AdAOH suppressed MCP-1 expression. In the absence of extracellular Mb, the iron-chelating drugs showed no material effect on MCP-1 secreted into the medium. The results represent the means±S.D. from three experiments. *Significantly different to the control; P<0.01. #Significantly different to the corresponding Mb-treated group; P<0.001.

Formation of stable nitroxide radicals

Enzymatic oxidation of DFOB by HRP is known to yield a stable nitroxide radical through hydrogen atom extraction from the hydroxamic acid moiety closest to the terminal amine group [35] (indicated by arrows in Figure 1). We recapitulated the generation of this radical using the HRP/H2O2 oxidizing system (results not shown) and demonstrated that a similar radical was formed in the reactions between DFOB-AdAOH and HRP/H2O2 as determined by EPR spectroscopy (Figure 7A). Simulation of the EPR spectrum indicated hyperfine couplings (aN 0.78 and aH 0.62 mT, obtained by spectral simulation to high correlations; r=0.98) (Figure 7B). The identical radical was formed in reactions of DFOB-AdAOH and Mb/H2O2 (Figure 7C). Consistent with HRP exhibiting greater peroxidase activity than Mb, the steady-state concentration of nitroxide radical was increased in mixtures containing HRP (compare Figures 7A and 7C). Notably, binding of Fe(III) to DFOB-AdAOH completely inhibited the formation of the nitroxide radical by Mb/H2O2 (Figure 7D), indicating that binding of iron regulated the availability of the hydroxamic acid moiety for enzymic oxidation. No radical products were detected in the absence of H2O2 or in mixtures of chelator and peroxide alone, indicating that an enzymatic reaction is required to yield the radical (results not shown).

Peroxidase enzymes oxidize DFOB or DFOB-AdAOH to yield a stable nitroxide radical

Figure 7
Peroxidase enzymes oxidize DFOB or DFOB-AdAOH to yield a stable nitroxide radical

Solutions of HRP (A) or Mb (C and D), both at a final concentration of 250 μM, were mixed with the chelator DFOB-AdAOH (final concentration 250 μM) either without (A and C) or with (D) ligation to Fe(III). Next, the reaction mixtures were treated with 500 μM H2O2 and incubated at 20°C for 2 min before transfer to a flat cell for EPR spectroscopy as described in the Experimental section. Spectrometer settings: power 100 mW, modulation amplitude 1 mT and sweep time 84 s. EPR spectra were recorded as an average of four cumulative scans. The spectrum shown in (B) represents EPR simulation of the spectrum in (A). Simulations were performed using WIMSIM software as described in the Experimental section. The results are representative of three independent experiments.

Figure 7
Peroxidase enzymes oxidize DFOB or DFOB-AdAOH to yield a stable nitroxide radical

Solutions of HRP (A) or Mb (C and D), both at a final concentration of 250 μM, were mixed with the chelator DFOB-AdAOH (final concentration 250 μM) either without (A and C) or with (D) ligation to Fe(III). Next, the reaction mixtures were treated with 500 μM H2O2 and incubated at 20°C for 2 min before transfer to a flat cell for EPR spectroscopy as described in the Experimental section. Spectrometer settings: power 100 mW, modulation amplitude 1 mT and sweep time 84 s. EPR spectra were recorded as an average of four cumulative scans. The spectrum shown in (B) represents EPR simulation of the spectrum in (A). Simulations were performed using WIMSIM software as described in the Experimental section. The results are representative of three independent experiments.

Addition of peroxide to mixtures containing Mb and DFOB-AdAOH resulted in near complete depletion of the chelator (99.0±3%: mean±S.D.; n=5) (Figure 8). No product peaks were detected in the period monitored by HPLC (up to 40 min) (results not shown). A similar extent of chelator depletion occurred with reaction mixtures containing Mb/H2O2 and DFOB (results not shown). Taken together, these results indicate that both DFOB-AdAOH and parental DFOB scavenge oxidants produced by peroxidase enzymes (including Mb, which has limited peroxidase ability [27]) in a redox reaction that involves generation of a free nitroxide radical.

Oxidation of DFOB-AdAOH with Mb completely depletes the chelator from solution

Figure 8
Oxidation of DFOB-AdAOH with Mb completely depletes the chelator from solution

Solutions of Mb (final concentration 250 μM) were mixed with DFOB-AdAOH (final concentration 250 μM) in the absence (A) or presence (B) of H2O2 (final concentration 500 μM). Next, the mixtures were incubated at 20°C for 5 min before analysis with reversed-phase HPLC. The arrow in (A) denotes the retention time for DFOB-AdAOH. Chromatograms shown are representative of two independent experiments each performed in triplicate.

Figure 8
Oxidation of DFOB-AdAOH with Mb completely depletes the chelator from solution

Solutions of Mb (final concentration 250 μM) were mixed with DFOB-AdAOH (final concentration 250 μM) in the absence (A) or presence (B) of H2O2 (final concentration 500 μM). Next, the mixtures were incubated at 20°C for 5 min before analysis with reversed-phase HPLC. The arrow in (A) denotes the retention time for DFOB-AdAOH. Chromatograms shown are representative of two independent experiments each performed in triplicate.

DISCUSSION

Multiple mechanisms have been suggested to explain the role of Mb in promoting acute renal injury following RM [13]. However, the exact link between the presence of extracellular Mb in the vascular system and acute kidney injury is not yet clear. The protective effect of DFOB in animal models of RM [15,16] has led to the notion that free iron plays a critical role in this acute pathology by participating in reactions that generate free radicals that cause an increase in cellular oxidative stress. Consequently, chelators that effectively bind and export iron from within cells have the potential to diminish intracellular oxidative stress and thereby protect renal cells from the effects of extracellular Mb and improve kidney function.

To attenuate the production of Fe(II)-dependent Fenton-based ROS, an ideal chelator will bind Fe as a stable Fe(III)–chelator complex that does not redox cycle to the Fe(II)–complex, since the dissociation of the latter could add to the Fe(II) burden. The thermodynamic stability of Fe(III)-loaded DFOB manifests as a negative Fe(III)/Fe(II) redox potential [E½=−0.48 V compared with NHE (normal hydrogen electrode) at pH 7.5] [36,37]. Since the structural modification in DFOB-AdAOH occurs in a region distal to that of Fe(III) co-ordination, the stability of Fe(III)-loaded DFOB-AdAOH will reflect closely that of Fe(III)-loaded DFOB. Indeed, previous work that studied the co-ordination chemistry of DFOB-AdAOH and analogues found that all complexes were formed as Fe(III) complexes and there was no evidence of Fe(II) co-ordination [24]. The potential value of DFOB-AdAOH above DFOB as a stable Fe(III)-based chelator lies in its reduced toxicity and its improved membrane partition properties, as reflected by logP values (DFOB-AdAOH, logP=0.11; DFOB, logP=−2.10) [24].

Consistent with the notion that extracellular Mb promotes renal epithelial dysfunction through enhancing oxidative stress [25,29], exposure of MDCK II cells to Mb in the presence of DHR-123 increased ROS production. This resulted in a parallel increase in the gene response for HO-1 and TNFα, implying an oxidative activation of the cells upon exposure to extracellular Mb. Pre-treatment of the cells with iron chelators decreased R-123 mean fluorescence intensity, indicating that the chelating agents diminished oxidative stress induced by Mb. Completely congruent with a decrease in oxidative stress, the gene response for HO-1 and pro-inflammatory TNFα also diminished to baseline levels upon pre-treatment with the chelators, with similar activities noted for both DFOB or DFOB-AdAOH.

On the basis of the results shown in the present study, it is clear that the chelators ameliorate Mb-induced oxidative stress in cultured kidney epithelial cells to similar extents. Gene expression of HO-1 can be dependent on antioxidant response elements in the promoter region of the gene encoding HO-1 [38], and the redox-sensitive transcription factor NF-κB is also linked to TNFα expression [39]. Whether this potential renal protective activity is due to the direct chelation of intracellular iron or the ability of the chelators to act as antioxidants, which inhibit Mb-oxidant production independent of iron chelation in the extracellular milieu [35,40], or alternatively a combination of these mechanisms, is not clear.

The unregulated pro-oxidant activity of extracellular Mb can lead to indiscriminate damage to lipids [41] and proteins [42], whereas redox cycling of Mb has been implicated in the pathogenesis of RM-induced renal failure [43]. We have shown previously that haptoglobin efficiently binds extracellular Mb and inhibits Mb-mediated renal cell dysfunction [25]. This renal-protective action can also be recapitulated by supplementing cells with a phenolic antioxidant [29]. Our results from the present study demonstrating that peroxidase enzymes are able to oxidize the iron-free chelators to yield a nitroxide radical indicate a potential for the chelators to inhibit the peroxidase activity of Mb by an oxidant-scavenging mechanism in addition to decreasing the impact of intracellular iron (over)load, effectively expanding their spectrum of biological activity.

Previous studies have suggested that free radicals can injure the renal tubular epithelium by initiating a cascade of pro-inflammatory mediators such as cytokines and chemokines [44]. For example, activation of the NF-κB pathway stimulates TNFα expression that in turn enhances apoptosis [45], increases glomerular damage and cytotoxicity and decreases renal cell viability [45]. In the case of renal epithelial cells, extracellular Mb elicits an inflammatory response through activation of the c-Src kinase-activator protein-1 and NF-κB pathways [14]. As a consequence, the activated or injured kidney cells secrete selectins, integrins and adhesion proteins [46]. Consistent with DFOB or DFOB-AdAOH exhibiting an anti-inflammatory action, both chelators down-regulated the expression of TNFα and subsequent secretion of MCP-1 in the presence of Mb, whereas in the absence of Mb the chelators had no impact on TNFα or MCP-1.

This anti-inflammatory activity may be beneficial to renal tissues during ARF. Secreted CCL2/MCP-1 plays a key role in acute and chronic renal diseases and leads to the infiltration and activation of monocytes [47], a hallmark of renal injury, and fibrosis in diabetic nephropathy [48]. The ability of iron chelators to decrease pro-inflammatory signalling in the kidney following RM probably results in a suppression of monocyte-macrophage activation driven by CCL2/MCP-1 as demonstrated in atherosclerosis [49].

Receptor-mediated endocytosis plays a crucial role in membranous transport function within kidney tubules. The decrease in transferrin uptake (shown in Figures 3 and 4) highlights the potential for extracellular Mb to promote the renal insufficiency that underlies ARF [50]. Transferrin receptors at the cell surface are sensitive to oxidative stress, presumably induced by thiol oxidation [31]. Humans do not have an active mechanism for iron excretion and the balance of intracellular iron is tightly regulated by the rate of erythropoiesis and distribution between intracellular iron stores by specialized proteins [51]. Therefore, a decrease in transferrin receptors can be interpreted as an overload of cellular iron, reflecting iron dysregulation. Importantly, both iron chelators increased transferrin uptake primarily through restoring the recycling of transferrin receptors to the cell surface. Whether this is due to an inhibition in oxidative stress as suggested previously [31] or whether chelation and removal of cellular iron plays a primary role in maintaining intracellular homoeostasis is presently unclear and further studies are warranted to elucidate which pathway is responsible for maintaining receptor-mediated endocytosis in this cell model of RM.

As well as receptor-specific transport via endocytosis, passive flow of ions/solutes following a concentration gradient is one of the major transport pathways in tubular epithelium [52]. Oxidative stress is known to impair epithelial function, potentially via damaging of tight junctions by free radicals and hydrogen peroxide acting as a protein tyrosine phosphatase inhibitor [53]. The supplementation of iron chelators significantly enhanced monolayer permeability in the presence of extracellular Mb, indicating a sustained tight junction function in the presence of Mb insult.

In this in vitro model of RM, supplementation with the iron chelator DFOB and the novel derivative DFOB-AdAOH resulted in overall enhanced cell function. We were able to demonstrate that iron chelators ameliorate oxidative stress and restore receptor-mediated transport as well as passive transport by maintaining epithelial monolayer permissiveness. Pre-treatment with the chelators also decreased the pro-inflammatory response elicited by extracellular Mb, and the pseudo-peroxidase was able to oxidize the chelators to yield the identical nitroxide radical. DFOB-AdAOH shows similar properties to DFOB. However, due to the decreased renal toxicity of this class of chelator [24], DFOB-AdAOH and other conjugated analogues show significant promise in the treatment of iron overload disease as well as a potential therapeutic strategy to combat ARF after RM.

Abbreviations

     
  • ARF

    acute renal failure

  •  
  • CCL2

    CC chemokine ligand 2

  •  
  • DFOB

    desferrioxamine B

  •  
  • DFOB-AdAOH

    DFOB-N-(3-hydroxyadamant-1-yl)carboxamide

  •  
  • DHR-123

    dihydrorhodamine-123

  •  
  • HO

    haem oxygenase

  •  
  • HRP

    horseradish peroxidase

  •  
  • ICAM

    intercellular adhesion molecule

  •  
  • MCP-1

    monocyte chemotactic protein 1

  •  
  • MDCK II cell

    Madin–Darby canine kidney II cell

  •  
  • Mb

    myoglobin

  •  
  • NF-κB

    nuclear factor κB

  •  
  • R-123

    rhodamine-123

  •  
  • RM

    rhabdomyolysis

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    reverse transcription

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • VCAM

    vascular cell adhesion molecule

AUTHOR CONTRIBUTION

Ludwig Groebler is a Visiting Research Trainee from Germany who is completing a doctorate while working in Paul Witting's laboratory. He was responsible for carrying out the majority of the work in the present study with guidance from the senior researcher. Together with Paul Witting, Ludwig Groebler was involved in formulating a draft manuscript. Anu Shanu is a Ph.D. student under the supervision of Paul Witting. She performed additional studies requested by the reviewers following assessment of the first submission of the manuscript. Her work investigated the impact of chelator supplementation on monolayer permissiveness and cellular production of MCP-1. Joe Liu is a Ph.D. student under the supervision of Rachel Codd. He was responsible for the preparation and purification of the chelator DFOB-AdAOH and its iron-bound form, and performing mass analyses. Joe Lui and Rachel Codd advised the research team on the use of the chelators and reviewed the results generated. All authors were involved in reviewing and updating the text associated with the revised manuscript.

FUNDING

This work was supported by the Australian Research Council [grant number DP0878559 (to P.K.W.)], the NHMRC (National Health and Medical Research Council) [Project 570844 (to R.C.)], The University of Sydney (Medical Faculty Strategic Research grant to R.C.) and the Bosch Molecular Biology Facility. L.K.G. is a visiting researcher from Berlin working in the Redox Biology Group. J.L. was awarded a co-funded Postgraduate Scholarship from the Faculty of Medicine at The University of Sydney.

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