Mol Imaging Biol (2015) DOI: 10.1007/s11307-015-0829-5 * World Molecular Imaging Society, 2015
RESEARCH ARTICLE
LOX-1-Targeted Iron Oxide Nanoparticles Detect Early Diabetic Nephropathy in db/db Mice Bing Luo,1 Song Wen,1 Yu-Chen Chen,1 Ying Cui,1 Fa-Bao Gao,2 Yu-Yu Yao,1 Sheng-Hong Ju,1 Gao-Jun Teng1 1
Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, 87 Dingjiaqiao Road, Nanjing, Jiangsu, 210009, China 2 Department of Radiology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
Abstract Purpose: Activation of the low-density lipoprotein receptor 1 (LOX-1) contributes to pervasive inflammation in early diabetic nephropathy (DN). This study determined the feasibility of antiLOX-1-ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) for noninvasive detection of inflammatory renal lesions in early DN. Procedures: Anti-mouse LOX-1 antibody was conjugated to polyethyleneglycol-coated USPIOs. In vitro analysis of USPIOs uptake was performed in RAW264.7 macrophages. DN and control mice were imaged by MRI prior to and 24 h after contrast treatment. Results: Anti-LOX-1 USPIOs were selectively taken up by macrophages, and kidney T2* MRI showed a lower signal intensity in the cortex of DN mice after 24 h administration of anti-LOX-1 USPIOs. Positive Perl’s staining in DN lesions, indicating the presence of iron oxide, was consistent with immunohistochemistry indicating the presence of LOX-1 and CD68. Conclusions: This report shows that anti-LOX-1 USPIOs detect LOX-1-enriched inflammatory renal lesions in early DN mice. Our study provides important information for characterizing and monitoring early DN. Key words: Diabetic nephropathy, Magnetic resonance imaging, LOX-1, USPIO
Background
D
iabetic nephropathy (DN) is a leading cause of endstage renal failure worldwide [1, 2] and constitutes a major component of progressive kidney disease. Due to its occult clinical presentation, DN is not usually diagnosed
Bing Luo and Song Wen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11307-015-0829-5) contains supplementary material, which is available to authorized users. Correspondence to: Gao-Jun Teng; e-mail: [email protected]
until late in its course, when most of the causal events have already played out and options for therapeutic intervention are constrained. Accordingly, early detection may enable the development of specific drugs and more timely initiation of therapy [3–5]. As reported by the Diabetes Control and Complications Trial/Epidemiology of Diabetes Complications (DCCT/EDIC) research group, patients who had undergone prior intensive therapy had substantially lower cumulative incidences of DN than those who had received conventional treatment, with less than 1 % requiring kidney replacement [6]. Focusing on markers of the early course of the disease might therefore facilitate the identification of DN and improve the likelihood of responsiveness to therapy.
B. Luo et al.: LOX-1-Targeted Iron Oxide Nanoparticles
Early nephropathy in both experimental and human diabetes is characterized by pervasive inflammation, a critical component of which is activation of the oxidized low-density lipoprotein receptor 1, LOX-1, which mediates uptake of low-density lipoproteins [7–9]. LOX-1 is expressed in macrophages, vascular endothelial cells, fibroblasts, and platelets. Binding of LOX-1 to oxLDL [10], other oxidized lipids [8], and leukocytes [11] activates the inflammatory cascade, leading to the expression of matrix metalloproteinases (MMPs) and MCP-1, and culminating in cell apoptosis [12, 13], endothelial dysfunction, and vasculopathy [14]. Similarly, LOX-1 expression in the renal capillaries and tubules of diabetic rats has been shown to be accompanied by intense oxidative stress, leukocyte infiltration, depressed mitochondrial enzyme level and function, and peritubular fibrosis. Interestingly, injection of the rats with anti-LOX-1 antibody has been shown to abrogate these abnormalities [15]. Based on these observations, a noninvasive in vivo molecular imaging approach to identify LOX-1 might prove useful for the early diagnosis of DN. Magnetic resonance imaging (MRI) is the modality of choice for detecting pathologic kidney changes due to its extraordinarily high temporal and spatial resolution [16, 17] and, as such represents a potentially attractive option for in vivo characterization of DN. Based on ultrasmall superparamagnetic iron oxide (USPIOs)’s unique physical, chemical, and mechanical properties, USPIO have been used to successfully detect renal abnormalities including nephrotoxic nephritis, obstructive nephropathy, ischemic renal disease, and transplanted kidney [18–20]. USPIOs can induce signal loss and susceptibility artifacts on T2- and T2*weighted MR images. The reason is iron oxide nanoparticles can be passively taken up by all available macrophages. In the present study, we developed an anti-LOX-1 USPIO probe and investigated its potential application in in vivo imaging of early renal lesion in DN mice.
were diluted in 2 ml boric acid/borate buffer (pH 9, 0.2 M). EDC.HCl (10 mg dissolved in borate buffer) and Sulfo-NHS (5 mg dissolved in borate buffer) were then added to the particle solution, and the reaction was allowed to continue for 30 min with continuous mixing. Next, 100 μg anti-mouse LOX-1 mAb (dissolved in 100 μl phosphate-buffered saline (PBS), 0.1 M, pH 7.4) was added, followed by stirring for 3 h at room temperature. Then, conjugated USPIO nanoparticles were purified three times with PBS using a centrifugal filter device (300KD, Sartorius, Germany) and stored in PBS (0.1 M, pH 7.4) at 4 °C. Nonspecific mouse IgG2a conjugated USPIO was used as control.
Characterization of Conjugated USPIO USPIO morphological examination was characterized by TEM (JEOL100CX), and the particle sizes and size distributions were calculated using the Image-Pro Plus 5.0 using at least 300 particles. The hydrated particle sizes were characterized by dynamic light scattering (DLS) (90 Plus Particle Size Analyzer, Brookhaven Instruments). The magnetic properties of the USPIOs were measured using a vibrating sample magnetometer (VSM, Lakeshore 7407). The longitudinal (R1) and transverse (R2) relaxation rates were determined at five different concentrations (0.1– 0.5 mM Fe) using a MRI scanner (Philips Achieva 3.0 T) The following sequences were used: (1) Look Locker T1 mapping sequence (repetition time/echo time 3.8/1.9 ms, flip angle 7°); (2) Multi-Slice Multi-Echo T2 mapping sequence (repletion time 2500 ms, echo time 19–112 ms, 16 echoes, flip angle 180°). The relaxivity values were calculated as the slope associated with a linear fit of the iron oxide concentration (mmol/l Fe) versus R1 (mmol/s) or R2 (mmol/s). The antigenic specificity of the LOX1-targeted nanoparticles was evaluated using a LOX-1 mouse ELISA kit (R&D systems).
In vitro Analysis of USPIO Uptake
Polyethyleneglycol-coated USPIOs (Fe3O4 nanoparticles, average particle size 11.8±1.1 nm, using α, ω -dicarboxyl-terminated polyglycol as a coating agent) which were provided by the Laboratory of Colloid, Interface and Chemical Thermodynamics, Chinese Academy of Sciences (Beijing, China) were synthesized via a Bone-pot^ reaction [21, 22]. Monoclonal anti-mouse LOX-1 antibody (mAb, Rat IgG2a) was purchased from R&D systems (Minneapolis, USA). 1-Ethyl-3(dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and Nhydroxysulfosuccnimide sodium salt (Sulfo-NHS) were purchased from Medpep Co. (Shanghai, China). Human oxLDL was purchased from Biosynthesis Biotechnology Co. (Beijing, China).
The murine macrophage cell line RAW264.7 was used for evaluation of the extent of passive uptake of PEG-coated targeted and untargeted USPIOs in activated foam macrophages. Cells were maintained in culture dishes with Dulbecco’s Modified Eagle’s medium (DMEM) media (Gibco, Carlsbad, CA) containing 10 % (w/v) fetal bovine serum in a humidified atmosphere of 5 % CO2 in air at 37 °C. Cells between passages 4 and 6 were used in the uptake experiments. For uptake experiments, 1×106 RAW264.7 macrophages were plated in 12-well plates with DMEM containing 10 % FBS, after which human oxLDL (100 μg/ml) was added and incubated for 12 h at 37 °C. Wells were washed three times with fresh DMEM, and the macrophages were used in the following experiments. AntiLOX-1 USPIOs and untargeted USPIOs were then incubated with the macrophages for an additional 12 h at 37 °C. For the competitive inhibition group, 50 μg/ml anti-LOX-1 mAb in PBS was administered 2 h prior to the administration of anti-LOX-1 USPIOs. Internalized iron oxide particles were detected by Perl’s staining with nuclear fast red counterstaining.
Synthesis of LOX-1-Targeted USPIO
In Vitro MRI
Methods for the synthesis of targeted USPIOs have been well described [23]. Briefly, 10 mg of PEG-coated USPIO nanoparticles
After the incubation of various USPIO nanoparticles for 12 h at 37 °C, RAW264.7 macrophages were washed 3–5 times with PBS followed
Methods Materials
B. Luo et al.: LOX-1-Targeted Iron Oxide Nanoparticles
by fixation with 4 % paraformaldehyde. Then, 1×105 macrophages from different groups were pelleted in 1 % agarose gel. Contrastenhanced MRI of the cells were obtained at 7.0 T using a 61-mm birdcage coil and a rat cradle. For MRI, a Turbo-RARE T2-weighted sequence was used (repetition time/echo time=2500 ms/33 ms, slice= 5, number of averages=6, matrix=256×256).
Animal Protocol The experimental protocols used in this study were approved by the Animal Care Committee of Southeast University, Nanjing, China. Male C57BL/6 mice and C57BL/6 background db/db mice (6 weeks old) were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). All animals were kept on a 12/12 h light-dark cycle with standard food and water freely available for 6 weeks prior to imaging. A total of 30 animals including 10 normal mice and 20 db/db mice were selected and divided into 3 groups (10 mice per group) as follows: group 1, db/ db mice+anti-LOX-1 USPIOs; group 2, db/db mice+untargeted USPIOs; and group 3, normal mice+anti-LOX-1 USPIOs.
average=8) was used prior to and 24 h after USPIO administration. The total in vivo imaging time was less than 10 min.
Data Analysis For signal intensity measurement, three independent regions of interest were selected on each side of the renal cortex, and the average T2* signal intensity was reported. The relative signal intensities (rSI) were then normalized with reference to the muscle (SI muscle) to exclude signal intensity fluctuations related to variations in technical parameters between the two imaging sequences. Image measurements were obtained using Paravision 5.0 software by an independent reader who was blinded to the types of USPIOs and histologic analysis. Percent normalized enhancement (%NENH) describes the percent change in rSI between the images before injection and at 24 h post-USPIO. The following formula was used: NENH=[(rSI post−rSI pre)/rSI pre]×100 %, where rSI post is the rSI obtained after injection and rSI pre is the rSI obtained before USPIO administration.
Morphological Analysis and Immunohistochemistry Metabolic Phenotype of Animal Models All metabolic parameters were assessed in 12-week-old mice prior to MR imaging.
Fasting Blood Glucose LevelsFasting glucose levels were measured in blood samples taken from the tail tip using a diagnostic autoanalyzer (ONETOUCH Ultra Easy; Johnson & Johnson, USA). Serum Lipid Biochemical AnalysisBlood samples were collected by cardiac puncture and centrifuged at 3000 rpm for 15 min to obtain serum. The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were assayed using the enzymatic method. Serum samples were examined using a colorimetric assay kit (BIOSINO, Beijing, China). Renal Function MeasurementSerum creatinine, urinary creatinine, urinary protein, and blood urea nitrogen levels were measured using a colorimetric assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. Urine samples were collected from mice housed in metabolic cages for 24 h.
In Vivo MRI In vivo MRI was performed at 7.0 T using a 35-mm birdcage coil and mouse cradle. Animals were initially anesthetized with a 4 % isoflurane/air gas mixture delivered through a nose cone and maintained under anesthesia with a 1.5–2 % isoflurane/air gas mixture. MRI was performed prior to and 24 h after tail vein injection of 10 mg Fe/kg body weight USPIO over 1 min. For in vivo imaging, a FLASH T2* sequence (TR/TE=250 ms/5 ms, flip angle=30°, slice thickness=1 mm, slices=15, number of
Kidney sections (8 μm) were stained with hematoxylin-eosin to identify structures and distinguish the cell nuclei. Perl’s staining was used to determine the presence of USPIOs. Immunolabeling with anti-LOX-1 mAb (1:100; R&D systems, Minneapolis, USA) and anti-CD68 antibody (1:100; Biolegend, San Diego, CA) was subsequently performed to demonstrate the location of LOX-1 and macrophages in the renal cortex. The number of CD68 positive cells was counted in high-power fields (×400, 950 glomeruli per section). All images were reviewed under light microscope (BX53, Olympus, Japan) by an independent pathologist. Computer-assisted morphometry was performed using Image-Pro Plus 5.0.
Statistical Analysis Data are indicated as mean±standard deviation. All multiple comparisons were made by one-way ANOVA followed by a Bonferroni post hoc test. All statistical tests were performed using SPSS for Windows (Version 17.0; SPSS). Differences with pG0.05 were considered statistically significant.
Results Characterization of USPIO Nanoparticles TEM images (Fig. 1a) showed that the anti-LOX-1 USPIOs nanoparticles were well dispersed in PBS solution. AntiLOX-1 USPIOs and untargeted USPIOs had similar hydrated diameters (29.68±2.95 and 27.12±4.12 nm). The R2 and R1 relaxation rates of anti-LOX-1 USPIOs, untargeted USPIOs were 181.48±0.13, 5.35±0.64 and 192.32±0.96, 5.26±0.67, respectively. Particle hydrodynamic size did not change significantly after 4 weeks, indicating their high stability. Furthermore, anti-LOX-1 USPIOs exhibited stability in PBS at 4 ° C for up to 20 weeks (data not shown). The
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Fig. 1 Characterization of iron oxide nanoparticles. a Representative TEM image of anti-LOX-1 USPIOs. b The room temperature magnetization curve of anti-LOX-1 USPIOs and untargeted USPIOs. c ELISA showing the biological activity of anti-LOX-1 USPIOs and unconjugated USPIOs. Data are presented as mean±SD (n=3).
saturation magnetization values of anti-LOX-1 USPIOs and unconjugated USPIOs were 27.18 and 23.81 emu/g Fe at 25 °C, respectively (Fig. 1b). ELISA results showed that the anti-LOX-1-mAb conjugated with USPIO retained high biological activity, whereas the untargeted USPIOs and boiled anti-LOX-1 had negligible activity (Fig. 1c).
Cellular Uptake of USPIOs Next, we assessed USPIO uptake by RAW264.7 foam macrophages (Fig. 2). Perl’s staining showed that uptake of anti-LOX-1 USPIOs by RAW264.7 cells was significantly higher than that of untargeted USPIOs (Fig. 2a) or USPIOs (Fig. 2c). Preincubation of the cells with anti-LOX-1 antibody eliminated uptake of the anti-LOX-1 USPIO nanoparticles (Fig. 2b). Our results indicate that anti-LOX1 USPIOs are specifically taken up by RAW264.7 foam macrophages, and that this uptake is mediated by RAW264.7 cell membrane LOX-1.
than the other PEG-coated USPIO groups (Fig. 3). The difference is significant when compared with the LOX-1targeted USPIO group (pG0.05).
Metabolic Parameters in db/db mice Fasting Blood Glucose Levels As shown in Fig. 4(a), fasting blood glucose level was significantly elevated in db/ db mice compared with the control mice (pG0.05). Serum Lipid Biochemical Analysis The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were significantly higher in db/db mice than in control mice (Fig. 4, pG0.05). Renal Function Measurement Serum creatinine, urinary creatinine, urinary protein, and blood urea nitrogen levels were significantly higher in db/db mice compared with control mice (Fig. 4, pG0.05).
In Vitro MRI In Vivo MRI Studies In vitro cell T2-weighted MRI demonstrated that the activated macrophages incubated with LOX-1-targeted USPIO (100 μg/ml) show significantly lower signal intensity
One hundred seven matched pairs (pre- and 24 h postinjection) of kidney MR images (42 for the db/db mice+
Fig. 2 Macrophage uptake of USPIOs. Macrophages were preincubated with human oxLDL (50 μg/ml) for 2 h prior to addition of USPIO nanoparticles. For the competitive inhibition group, anti-LOX-1 monoclonal antibody (50 μg/ml) was preincubated with macrophages before addition of anti-LOX-1 USPIOs. a Perl’s staining shows highest uptake of nanoparticles in the antiLOX-1 USPIO group. b Uptake of targeted USPIO nanoparticles was eliminated by preincubation with anti-LOX-1 antibody. c Macrophages incubated with untargeted IgG-USPIO have limited USPIO uptake. Bar indicates 30 μm.
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Fig. 3 In vitro T2-weighted MRI. a T2 MR Images of activated macrophages labeled with various USPIOs for 12 h. b rSI is quantitatively compared for different groups. Asterisk indicates pG0.05 compared with LOX-1-targeted USPIO.
anti-LOX-1 USPIOs group, 35 for the db/db mice + untargeted USPIOs group, and 30 for the normal mice+ anti-LOX-1 USPIOs) were selected for comparative analysis. Compared with the preinjection MRIs, we observed a decrease (−33.26±8.72 %; pG0.05) in the T2*-weighted MRIs in the cortical compartment of db/db mice 24 h after anti-LOX-1 USPIO injection (Fig. 5). There was no significant difference in cortical signal intensity between images obtained prior to and 24 h after injection in either db/ db mice injected with untargeted USPIOs, or in the control group (Fig. 5). No toxicity was observed in any mouse group.
Deposition of LOX-1 Targeted USPIO in the Glomeruli Relative to the db/db untargeted USPIOs group and the antiLOX-1 USPIO control mouse group, Perl’s staining indicated significantly higher levels of iron in the glomeruli of animals in the db/db+anti-LOX-1 USPIOs group (Fig. 6). Consistent with this, immunolabeling with LOX-1 and CD68 antibodies showed significantly higher levels of LOX-1 and macrophages in the glomeruli of db/db than in the control mice. Moreover, there was a significant
correlation (r=0.736, pG0.0001) between the number of glomerular CD68-positive cells and the preinjection vs. 24 h post-injection difference in signal intensity in DN mice treated with anti-LOX-1 USPIOs.
Discussion In the previous study [24], we report a sensitive, specific, and biocompatible LOX-1-targeted USPIO for the noninvasive MR imaging of LOX-1 within carotid atherosclerotic lesions in apoE-deficient mice. In additional studies, we found the deposition of LOX-1-targeted USPIO in the glomerulus of apoE−/−mice, which was co-localized with macrophages and LOX-1 expression, suggesting that this targeted probe may have potential to noninvasively image glomerular disease. Therefore, in the present study, we have demonstrated the use of a specific anti-LOX-1 USPIOs MRI probe for noninvasive detection of LOX-1-enriched inflammatory renal lesions in a mouse model of early DN. Injection of anti-LOX-1 USPIOs in DN mice caused a significant reduction in T2*-weighted cortical MR signal intensity that was not observed in normal mice injected with anti-LOX-1 USPIOs, nor in DN mice injected with
B. Luo et al.: LOX-1-Targeted Iron Oxide Nanoparticles
Fig. 4 Metabolic parameters of db/db and control mice. a Fasting blood glucose level was significantly elevated in db/db mice compared with the control mice. b The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were significantly higher in db/db mice than in control mice. c Urinary protein, d urinary creatinine, e serum creatinine, and f blood urea nitrogen levels were significantly increased in db/db mice compared with control mice. (*pG0.05 when compared with normal mice). Data are presented as mean±SD.
Fig. 5 In vivo T2*-weighted images. aIn vivo T2* MRI demonstrated a reduction in signal in the glomerulus of early DN mice 24 h after administration of anti-LOX-1 USPIO nanoparticles (dwhite arrows indicate the location of signal loss within the plaque). There was limited signal reduction in the db/db mice+untargeted USPIOs group (e) and the normal mice+anti-LOX-1 USPIOs group (f). b Relative signal intensity changes (NENH%) in the T2*-weighted images associated with the renal cortex before and after administration of USPIOs (*pG0.05 when compared normal mice).
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Fig. 6 Histomorphometry and immunohistochemistry of USPIOs in the glomeruli of DN mice. a Positive staining for iron oxide, d LOX-1, and g macrophages in the glomeruli of db/db mice injected with anti-LOX-1 USPIOs (yellow arrow). Limited iron oxide deposition in the glomeruli of db/db mice injected with b untargeted USPIOs, although staining for e LOX-1 and h CD68 is positive. c Absence of iron oxide, f LOX-1, and i CD68 in the glomeruli of normal mice (Bar=100 μm).
untargeted USPIOs. The high image quality, sensitivity, and resolution of MRI, combined with the limited toxicity of the targeted USPIOs [25], suggest that the anti-LOX-1 USPIOs we have described represent a potentially valuable approach for the early detection and monitoring of DN. Recent studies have demonstrated that activation of inflammatory pathways plays a central role in the early stages of DN, leading to the activation and recruitment of fibroblasts and culminating in renal fibrosis [26]. LOX-1 expression in renal tissue has been shown to play a crucial role in inflammatory changes associated with DN [27–29]. While the basal expression of LOX-1 is low, it is highly induced by its ligand, oxLDL, in addition to inflammatory cytokines, including TNF-α, interleukin1β (IL-1β), transforming growth factor-β1(TGF-β1), as well as free fatty acids, angiotensin II, endothelin-1, hyperglycemia, and hemodynamic changes [8, 30–33]. Zhang et al. have proposed that in response to activation of the LOX-1 pathway by oxidized HDL, glomerular mesangial cells adopt a dysfunctional, proinflammatory state, ultimately succumbing to apoptosis [34]. Collectively, these data afforded us a sound rationale for leveraging LOX-1 as a biomarker for the detection of early renal lesions in DN. The anticipated symptoms of hyperglycemia, dyslipidemia, polyphagia, polydipsia, and polyuria were observed in the db/db mice. Moreover, as expected, levels of urinary creatinine, urinary
protein, serum lipid, serum creatinine, and blood urea nitrogen were significantly higher in 12-week-old db/db mice than in agematched nondiabetic mice, all of which are indicative of renal dysfunction. Based on these data, 12-week-old db/db mice had pathological characteristics comparable to the early stages of human DN [3], and were therefore considered appropriate models for the current study. The PEG coating on the anti-LOX-1 USPIOs reduces plasma protein binding, retards clearance by the reticuloendothelial system, and increases particle circulation times [35], all of which increased the probability of USPIOs reaching the tissue of interest [36]. In vitro experiments showed the PEG-coated USPIO nanoparticles were not passively taken up by RAW264.7 macrophages exposed to human oxLDL for 12 h. However, Perl’s staining demonstrated that LOX-1-targeted USPIO were taken up by activated macrophages cells due to elevated membrane expression levels of LOX-1 [37]. In contrast, uptake of anti-LOX-1 USPIOs was lower in macrophages not preincubated with oxLDL due to their lower membrane expression levels of LOX-1. Collectively, these results demonstrate that LOX-1targeted PEG-coated USPIO nanoparticles selectively accumulate in activated macrophages expressing LOX-1. After in vitro analysis of USPIO uptake, we next used MR imaging to demonstrate early detection of DN by the LOX-1-targeted USPIOs in vivo. Related studies have
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reported the noninvasive detection of inflammation in a rat model of ischemic acute renal failure by USPIOenhanced MRI, with no effect on renal injury or recovery [18]. Moreover Li et al. [25] developed a novel LOX-1 targeted molecular imaging probe that bound preferentially to the plaque shoulder of the aortic arch in atherosclerotic apoE-/- mice and LDLR-/- mice, a region with extensive LOX-1 expression and macrophage accumulation. These results indicated that in vivo imaging of LOX-1 expression using an LOX-1 antibody represented a potential approach to identifying inflammatory renal lesions in early DN. In the current study, DN mice and age-matched C57BL/6 mice were examined using a T2*weighted sequence. The MR images showed a significant signal intensity decrease after 24 h administration of anti-LOX-1 USPIO in the cortex of DN mice, but not normal mice injected with targeted USPIO, nor in DN mice injected with untargeted USPIO. Moreover, Perl’s staining revealed and indicated that the iron-containing cells were mainly located in the glomerulus in DN mice injected with LOX-1-targeted USPIO particles. Furthermore, immunohistochemistry and Western blotting analysis confirmed the iron-containing cells co-localized with LOX-1/macrophage expression. In addition, we found a significant negative correlation between the percent normalized enhancement and the number of CD68-positive macrophages. In concert, these data demonstrate that LOX-1-targeted USPIOs can be used for the molecular imaging of LOX-1 enriched DN kidney lesions in vivo. Macrophages are the principle inflammatory cells in the diabetic kidney and play a crucial role in the pathogenesis of glomerulopathy lesions in DN [38], and the results indicating that the signal reduction caused by administration of LOX-1 targeted USPIOs accurately reflects the inflammatory responses characteristic of early DN. Considering the limited cell toxicity of the USPIOs in the current study compared with Gdcontaining micelles, our method represents a clinically feasible method with which for the early detection and diagnosis of DN. The limitations of this study include its undetermined relevance to humans, and the fact that the USPIO dose (10 mg/kg) exceeds that recommended for clinical applications. This dose was chosen to increase the probability of USPIO uptake by macrophages, since the half-life of these particles in rats is shorter than in humans. A final limitation of our study is the lack of information on the safety of these nanoparticles in humans, which we will evaluate in future studies. In summary, the present study demonstrates a specific, sensitive method for noninvasive imaging LOX-1 within DN renal lesions, which can potentially provide important information for the early detection of DN. Due to the limited toxicity of the USPIOs compared with Gd-containing micelles, our method represents a novel potential clinical diagnostic modality for DN.
Sources of Funding. This work was supported by the Major State Basic Research Development Program of China (973 Program) (NOs. 2013CB733800, 2013CB733803), National Natural Science Foundation of China (NOs. 81230034, 81271739, 81271637, 81401460), and Jiangsu Provincial Special Program of Medical Science (BL2013029). Conflict of Interest. The authors have declared that no competing interests exist.
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21. Gao Q, Zhang J, Hong G, Ni J (2010) One-pot reaction to synthesize PEG-coated hollow magnetite nanostructures with excellent magnetic properties. J Nanosci Nanotechnol 10:6400–6406 22. Zhen L, Wei L, Gao MY et al (2005) One-pot reaction to synthesize biocompatible magnetite nanoparticles. Adv Mater 17:1001–1005 23. Wen S, Liu DF, Liu Z et al (2012) OxLDL-targeted iron oxide nanoparticles for in vivo MRI detection of perivascular carotid collar induced atherosclerotic lesions in ApoE-deficient mice. J Lipid Res 53:829–838 24. Wen S, Liu DF, Cui Y et al (2014) In vivo MRI detection of carotid atherosclerotic lesions and kidney inflammation in ApoE-deficient mice by using LOX-1 targeted iron nanoparticles. Nanomedicine 10:639–649 25. Li D, Patel AR, Klibanov AL et al (2010) Molecular imaging of atherosclerotic plaques targeted to oxidized LDL receptor LOX-1 by SPECT/CT and magnetic resonance. Circ Cardiovasc Imaging 3:464–472 26. Kanasaki K, Taduri G, Koya D (2013) Diabetic nephropathy: the role of inflammation in fibroblast activation and kidney fibrosis. Front Endocrinol (Lausanne) 4:7 27. Kenney WL, Cannon JG, Alexander LM (2013) Cutaneous microvascular dysfunction correlates with serum LDL and sLOX-1 receptor concentrations. Microvasc Res 85:112–117 28. Palmieri VO, Coppola B, Grattagliano I et al (2013) Oxidized LDL receptor 1 gene polymorphism in patients with metabolic syndrome. Eur J Clin Invest 43:41–48 29. Dominguez J, Wu P, Packer CS et al (2007) Lipotoxic and inflammatory phenotypes in rats with uncontrolled metabolic syndrome and nephropathy. Am J Physiol Renal Physiol 293:F670–F679 30. Lopes-Virella MF, Hunt KJ, Baker NL et al (2011) Levels of oxidized LDL and advanced glycation end products-modified LDL in circulating
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RESEARCH ARTICLE
LOX-1-Targeted Iron Oxide Nanoparticles Detect Early Diabetic Nephropathy in db/db Mice Bing Luo,1 Song Wen,1 Yu-Chen Chen,1 Ying Cui,1 Fa-Bao Gao,2 Yu-Yu Yao,1 Sheng-Hong Ju,1 Gao-Jun Teng1 1
Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, 87 Dingjiaqiao Road, Nanjing, Jiangsu, 210009, China 2 Department of Radiology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
Abstract Purpose: Activation of the low-density lipoprotein receptor 1 (LOX-1) contributes to pervasive inflammation in early diabetic nephropathy (DN). This study determined the feasibility of antiLOX-1-ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) for noninvasive detection of inflammatory renal lesions in early DN. Procedures: Anti-mouse LOX-1 antibody was conjugated to polyethyleneglycol-coated USPIOs. In vitro analysis of USPIOs uptake was performed in RAW264.7 macrophages. DN and control mice were imaged by MRI prior to and 24 h after contrast treatment. Results: Anti-LOX-1 USPIOs were selectively taken up by macrophages, and kidney T2* MRI showed a lower signal intensity in the cortex of DN mice after 24 h administration of anti-LOX-1 USPIOs. Positive Perl’s staining in DN lesions, indicating the presence of iron oxide, was consistent with immunohistochemistry indicating the presence of LOX-1 and CD68. Conclusions: This report shows that anti-LOX-1 USPIOs detect LOX-1-enriched inflammatory renal lesions in early DN mice. Our study provides important information for characterizing and monitoring early DN. Key words: Diabetic nephropathy, Magnetic resonance imaging, LOX-1, USPIO
Background
D
iabetic nephropathy (DN) is a leading cause of endstage renal failure worldwide [1, 2] and constitutes a major component of progressive kidney disease. Due to its occult clinical presentation, DN is not usually diagnosed
Bing Luo and Song Wen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11307-015-0829-5) contains supplementary material, which is available to authorized users. Correspondence to: Gao-Jun Teng; e-mail: [email protected]
until late in its course, when most of the causal events have already played out and options for therapeutic intervention are constrained. Accordingly, early detection may enable the development of specific drugs and more timely initiation of therapy [3–5]. As reported by the Diabetes Control and Complications Trial/Epidemiology of Diabetes Complications (DCCT/EDIC) research group, patients who had undergone prior intensive therapy had substantially lower cumulative incidences of DN than those who had received conventional treatment, with less than 1 % requiring kidney replacement [6]. Focusing on markers of the early course of the disease might therefore facilitate the identification of DN and improve the likelihood of responsiveness to therapy.
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Early nephropathy in both experimental and human diabetes is characterized by pervasive inflammation, a critical component of which is activation of the oxidized low-density lipoprotein receptor 1, LOX-1, which mediates uptake of low-density lipoproteins [7–9]. LOX-1 is expressed in macrophages, vascular endothelial cells, fibroblasts, and platelets. Binding of LOX-1 to oxLDL [10], other oxidized lipids [8], and leukocytes [11] activates the inflammatory cascade, leading to the expression of matrix metalloproteinases (MMPs) and MCP-1, and culminating in cell apoptosis [12, 13], endothelial dysfunction, and vasculopathy [14]. Similarly, LOX-1 expression in the renal capillaries and tubules of diabetic rats has been shown to be accompanied by intense oxidative stress, leukocyte infiltration, depressed mitochondrial enzyme level and function, and peritubular fibrosis. Interestingly, injection of the rats with anti-LOX-1 antibody has been shown to abrogate these abnormalities [15]. Based on these observations, a noninvasive in vivo molecular imaging approach to identify LOX-1 might prove useful for the early diagnosis of DN. Magnetic resonance imaging (MRI) is the modality of choice for detecting pathologic kidney changes due to its extraordinarily high temporal and spatial resolution [16, 17] and, as such represents a potentially attractive option for in vivo characterization of DN. Based on ultrasmall superparamagnetic iron oxide (USPIOs)’s unique physical, chemical, and mechanical properties, USPIO have been used to successfully detect renal abnormalities including nephrotoxic nephritis, obstructive nephropathy, ischemic renal disease, and transplanted kidney [18–20]. USPIOs can induce signal loss and susceptibility artifacts on T2- and T2*weighted MR images. The reason is iron oxide nanoparticles can be passively taken up by all available macrophages. In the present study, we developed an anti-LOX-1 USPIO probe and investigated its potential application in in vivo imaging of early renal lesion in DN mice.
were diluted in 2 ml boric acid/borate buffer (pH 9, 0.2 M). EDC.HCl (10 mg dissolved in borate buffer) and Sulfo-NHS (5 mg dissolved in borate buffer) were then added to the particle solution, and the reaction was allowed to continue for 30 min with continuous mixing. Next, 100 μg anti-mouse LOX-1 mAb (dissolved in 100 μl phosphate-buffered saline (PBS), 0.1 M, pH 7.4) was added, followed by stirring for 3 h at room temperature. Then, conjugated USPIO nanoparticles were purified three times with PBS using a centrifugal filter device (300KD, Sartorius, Germany) and stored in PBS (0.1 M, pH 7.4) at 4 °C. Nonspecific mouse IgG2a conjugated USPIO was used as control.
Characterization of Conjugated USPIO USPIO morphological examination was characterized by TEM (JEOL100CX), and the particle sizes and size distributions were calculated using the Image-Pro Plus 5.0 using at least 300 particles. The hydrated particle sizes were characterized by dynamic light scattering (DLS) (90 Plus Particle Size Analyzer, Brookhaven Instruments). The magnetic properties of the USPIOs were measured using a vibrating sample magnetometer (VSM, Lakeshore 7407). The longitudinal (R1) and transverse (R2) relaxation rates were determined at five different concentrations (0.1– 0.5 mM Fe) using a MRI scanner (Philips Achieva 3.0 T) The following sequences were used: (1) Look Locker T1 mapping sequence (repetition time/echo time 3.8/1.9 ms, flip angle 7°); (2) Multi-Slice Multi-Echo T2 mapping sequence (repletion time 2500 ms, echo time 19–112 ms, 16 echoes, flip angle 180°). The relaxivity values were calculated as the slope associated with a linear fit of the iron oxide concentration (mmol/l Fe) versus R1 (mmol/s) or R2 (mmol/s). The antigenic specificity of the LOX1-targeted nanoparticles was evaluated using a LOX-1 mouse ELISA kit (R&D systems).
In vitro Analysis of USPIO Uptake
Polyethyleneglycol-coated USPIOs (Fe3O4 nanoparticles, average particle size 11.8±1.1 nm, using α, ω -dicarboxyl-terminated polyglycol as a coating agent) which were provided by the Laboratory of Colloid, Interface and Chemical Thermodynamics, Chinese Academy of Sciences (Beijing, China) were synthesized via a Bone-pot^ reaction [21, 22]. Monoclonal anti-mouse LOX-1 antibody (mAb, Rat IgG2a) was purchased from R&D systems (Minneapolis, USA). 1-Ethyl-3(dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and Nhydroxysulfosuccnimide sodium salt (Sulfo-NHS) were purchased from Medpep Co. (Shanghai, China). Human oxLDL was purchased from Biosynthesis Biotechnology Co. (Beijing, China).
The murine macrophage cell line RAW264.7 was used for evaluation of the extent of passive uptake of PEG-coated targeted and untargeted USPIOs in activated foam macrophages. Cells were maintained in culture dishes with Dulbecco’s Modified Eagle’s medium (DMEM) media (Gibco, Carlsbad, CA) containing 10 % (w/v) fetal bovine serum in a humidified atmosphere of 5 % CO2 in air at 37 °C. Cells between passages 4 and 6 were used in the uptake experiments. For uptake experiments, 1×106 RAW264.7 macrophages were plated in 12-well plates with DMEM containing 10 % FBS, after which human oxLDL (100 μg/ml) was added and incubated for 12 h at 37 °C. Wells were washed three times with fresh DMEM, and the macrophages were used in the following experiments. AntiLOX-1 USPIOs and untargeted USPIOs were then incubated with the macrophages for an additional 12 h at 37 °C. For the competitive inhibition group, 50 μg/ml anti-LOX-1 mAb in PBS was administered 2 h prior to the administration of anti-LOX-1 USPIOs. Internalized iron oxide particles were detected by Perl’s staining with nuclear fast red counterstaining.
Synthesis of LOX-1-Targeted USPIO
In Vitro MRI
Methods for the synthesis of targeted USPIOs have been well described [23]. Briefly, 10 mg of PEG-coated USPIO nanoparticles
After the incubation of various USPIO nanoparticles for 12 h at 37 °C, RAW264.7 macrophages were washed 3–5 times with PBS followed
Methods Materials
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by fixation with 4 % paraformaldehyde. Then, 1×105 macrophages from different groups were pelleted in 1 % agarose gel. Contrastenhanced MRI of the cells were obtained at 7.0 T using a 61-mm birdcage coil and a rat cradle. For MRI, a Turbo-RARE T2-weighted sequence was used (repetition time/echo time=2500 ms/33 ms, slice= 5, number of averages=6, matrix=256×256).
Animal Protocol The experimental protocols used in this study were approved by the Animal Care Committee of Southeast University, Nanjing, China. Male C57BL/6 mice and C57BL/6 background db/db mice (6 weeks old) were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). All animals were kept on a 12/12 h light-dark cycle with standard food and water freely available for 6 weeks prior to imaging. A total of 30 animals including 10 normal mice and 20 db/db mice were selected and divided into 3 groups (10 mice per group) as follows: group 1, db/ db mice+anti-LOX-1 USPIOs; group 2, db/db mice+untargeted USPIOs; and group 3, normal mice+anti-LOX-1 USPIOs.
average=8) was used prior to and 24 h after USPIO administration. The total in vivo imaging time was less than 10 min.
Data Analysis For signal intensity measurement, three independent regions of interest were selected on each side of the renal cortex, and the average T2* signal intensity was reported. The relative signal intensities (rSI) were then normalized with reference to the muscle (SI muscle) to exclude signal intensity fluctuations related to variations in technical parameters between the two imaging sequences. Image measurements were obtained using Paravision 5.0 software by an independent reader who was blinded to the types of USPIOs and histologic analysis. Percent normalized enhancement (%NENH) describes the percent change in rSI between the images before injection and at 24 h post-USPIO. The following formula was used: NENH=[(rSI post−rSI pre)/rSI pre]×100 %, where rSI post is the rSI obtained after injection and rSI pre is the rSI obtained before USPIO administration.
Morphological Analysis and Immunohistochemistry Metabolic Phenotype of Animal Models All metabolic parameters were assessed in 12-week-old mice prior to MR imaging.
Fasting Blood Glucose LevelsFasting glucose levels were measured in blood samples taken from the tail tip using a diagnostic autoanalyzer (ONETOUCH Ultra Easy; Johnson & Johnson, USA). Serum Lipid Biochemical AnalysisBlood samples were collected by cardiac puncture and centrifuged at 3000 rpm for 15 min to obtain serum. The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were assayed using the enzymatic method. Serum samples were examined using a colorimetric assay kit (BIOSINO, Beijing, China). Renal Function MeasurementSerum creatinine, urinary creatinine, urinary protein, and blood urea nitrogen levels were measured using a colorimetric assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. Urine samples were collected from mice housed in metabolic cages for 24 h.
In Vivo MRI In vivo MRI was performed at 7.0 T using a 35-mm birdcage coil and mouse cradle. Animals were initially anesthetized with a 4 % isoflurane/air gas mixture delivered through a nose cone and maintained under anesthesia with a 1.5–2 % isoflurane/air gas mixture. MRI was performed prior to and 24 h after tail vein injection of 10 mg Fe/kg body weight USPIO over 1 min. For in vivo imaging, a FLASH T2* sequence (TR/TE=250 ms/5 ms, flip angle=30°, slice thickness=1 mm, slices=15, number of
Kidney sections (8 μm) were stained with hematoxylin-eosin to identify structures and distinguish the cell nuclei. Perl’s staining was used to determine the presence of USPIOs. Immunolabeling with anti-LOX-1 mAb (1:100; R&D systems, Minneapolis, USA) and anti-CD68 antibody (1:100; Biolegend, San Diego, CA) was subsequently performed to demonstrate the location of LOX-1 and macrophages in the renal cortex. The number of CD68 positive cells was counted in high-power fields (×400, 950 glomeruli per section). All images were reviewed under light microscope (BX53, Olympus, Japan) by an independent pathologist. Computer-assisted morphometry was performed using Image-Pro Plus 5.0.
Statistical Analysis Data are indicated as mean±standard deviation. All multiple comparisons were made by one-way ANOVA followed by a Bonferroni post hoc test. All statistical tests were performed using SPSS for Windows (Version 17.0; SPSS). Differences with pG0.05 were considered statistically significant.
Results Characterization of USPIO Nanoparticles TEM images (Fig. 1a) showed that the anti-LOX-1 USPIOs nanoparticles were well dispersed in PBS solution. AntiLOX-1 USPIOs and untargeted USPIOs had similar hydrated diameters (29.68±2.95 and 27.12±4.12 nm). The R2 and R1 relaxation rates of anti-LOX-1 USPIOs, untargeted USPIOs were 181.48±0.13, 5.35±0.64 and 192.32±0.96, 5.26±0.67, respectively. Particle hydrodynamic size did not change significantly after 4 weeks, indicating their high stability. Furthermore, anti-LOX-1 USPIOs exhibited stability in PBS at 4 ° C for up to 20 weeks (data not shown). The
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Fig. 1 Characterization of iron oxide nanoparticles. a Representative TEM image of anti-LOX-1 USPIOs. b The room temperature magnetization curve of anti-LOX-1 USPIOs and untargeted USPIOs. c ELISA showing the biological activity of anti-LOX-1 USPIOs and unconjugated USPIOs. Data are presented as mean±SD (n=3).
saturation magnetization values of anti-LOX-1 USPIOs and unconjugated USPIOs were 27.18 and 23.81 emu/g Fe at 25 °C, respectively (Fig. 1b). ELISA results showed that the anti-LOX-1-mAb conjugated with USPIO retained high biological activity, whereas the untargeted USPIOs and boiled anti-LOX-1 had negligible activity (Fig. 1c).
Cellular Uptake of USPIOs Next, we assessed USPIO uptake by RAW264.7 foam macrophages (Fig. 2). Perl’s staining showed that uptake of anti-LOX-1 USPIOs by RAW264.7 cells was significantly higher than that of untargeted USPIOs (Fig. 2a) or USPIOs (Fig. 2c). Preincubation of the cells with anti-LOX-1 antibody eliminated uptake of the anti-LOX-1 USPIO nanoparticles (Fig. 2b). Our results indicate that anti-LOX1 USPIOs are specifically taken up by RAW264.7 foam macrophages, and that this uptake is mediated by RAW264.7 cell membrane LOX-1.
than the other PEG-coated USPIO groups (Fig. 3). The difference is significant when compared with the LOX-1targeted USPIO group (pG0.05).
Metabolic Parameters in db/db mice Fasting Blood Glucose Levels As shown in Fig. 4(a), fasting blood glucose level was significantly elevated in db/ db mice compared with the control mice (pG0.05). Serum Lipid Biochemical Analysis The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were significantly higher in db/db mice than in control mice (Fig. 4, pG0.05). Renal Function Measurement Serum creatinine, urinary creatinine, urinary protein, and blood urea nitrogen levels were significantly higher in db/db mice compared with control mice (Fig. 4, pG0.05).
In Vitro MRI In Vivo MRI Studies In vitro cell T2-weighted MRI demonstrated that the activated macrophages incubated with LOX-1-targeted USPIO (100 μg/ml) show significantly lower signal intensity
One hundred seven matched pairs (pre- and 24 h postinjection) of kidney MR images (42 for the db/db mice+
Fig. 2 Macrophage uptake of USPIOs. Macrophages were preincubated with human oxLDL (50 μg/ml) for 2 h prior to addition of USPIO nanoparticles. For the competitive inhibition group, anti-LOX-1 monoclonal antibody (50 μg/ml) was preincubated with macrophages before addition of anti-LOX-1 USPIOs. a Perl’s staining shows highest uptake of nanoparticles in the antiLOX-1 USPIO group. b Uptake of targeted USPIO nanoparticles was eliminated by preincubation with anti-LOX-1 antibody. c Macrophages incubated with untargeted IgG-USPIO have limited USPIO uptake. Bar indicates 30 μm.
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Fig. 3 In vitro T2-weighted MRI. a T2 MR Images of activated macrophages labeled with various USPIOs for 12 h. b rSI is quantitatively compared for different groups. Asterisk indicates pG0.05 compared with LOX-1-targeted USPIO.
anti-LOX-1 USPIOs group, 35 for the db/db mice + untargeted USPIOs group, and 30 for the normal mice+ anti-LOX-1 USPIOs) were selected for comparative analysis. Compared with the preinjection MRIs, we observed a decrease (−33.26±8.72 %; pG0.05) in the T2*-weighted MRIs in the cortical compartment of db/db mice 24 h after anti-LOX-1 USPIO injection (Fig. 5). There was no significant difference in cortical signal intensity between images obtained prior to and 24 h after injection in either db/ db mice injected with untargeted USPIOs, or in the control group (Fig. 5). No toxicity was observed in any mouse group.
Deposition of LOX-1 Targeted USPIO in the Glomeruli Relative to the db/db untargeted USPIOs group and the antiLOX-1 USPIO control mouse group, Perl’s staining indicated significantly higher levels of iron in the glomeruli of animals in the db/db+anti-LOX-1 USPIOs group (Fig. 6). Consistent with this, immunolabeling with LOX-1 and CD68 antibodies showed significantly higher levels of LOX-1 and macrophages in the glomeruli of db/db than in the control mice. Moreover, there was a significant
correlation (r=0.736, pG0.0001) between the number of glomerular CD68-positive cells and the preinjection vs. 24 h post-injection difference in signal intensity in DN mice treated with anti-LOX-1 USPIOs.
Discussion In the previous study [24], we report a sensitive, specific, and biocompatible LOX-1-targeted USPIO for the noninvasive MR imaging of LOX-1 within carotid atherosclerotic lesions in apoE-deficient mice. In additional studies, we found the deposition of LOX-1-targeted USPIO in the glomerulus of apoE−/−mice, which was co-localized with macrophages and LOX-1 expression, suggesting that this targeted probe may have potential to noninvasively image glomerular disease. Therefore, in the present study, we have demonstrated the use of a specific anti-LOX-1 USPIOs MRI probe for noninvasive detection of LOX-1-enriched inflammatory renal lesions in a mouse model of early DN. Injection of anti-LOX-1 USPIOs in DN mice caused a significant reduction in T2*-weighted cortical MR signal intensity that was not observed in normal mice injected with anti-LOX-1 USPIOs, nor in DN mice injected with
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Fig. 4 Metabolic parameters of db/db and control mice. a Fasting blood glucose level was significantly elevated in db/db mice compared with the control mice. b The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in serum were significantly higher in db/db mice than in control mice. c Urinary protein, d urinary creatinine, e serum creatinine, and f blood urea nitrogen levels were significantly increased in db/db mice compared with control mice. (*pG0.05 when compared with normal mice). Data are presented as mean±SD.
Fig. 5 In vivo T2*-weighted images. aIn vivo T2* MRI demonstrated a reduction in signal in the glomerulus of early DN mice 24 h after administration of anti-LOX-1 USPIO nanoparticles (dwhite arrows indicate the location of signal loss within the plaque). There was limited signal reduction in the db/db mice+untargeted USPIOs group (e) and the normal mice+anti-LOX-1 USPIOs group (f). b Relative signal intensity changes (NENH%) in the T2*-weighted images associated with the renal cortex before and after administration of USPIOs (*pG0.05 when compared normal mice).
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Fig. 6 Histomorphometry and immunohistochemistry of USPIOs in the glomeruli of DN mice. a Positive staining for iron oxide, d LOX-1, and g macrophages in the glomeruli of db/db mice injected with anti-LOX-1 USPIOs (yellow arrow). Limited iron oxide deposition in the glomeruli of db/db mice injected with b untargeted USPIOs, although staining for e LOX-1 and h CD68 is positive. c Absence of iron oxide, f LOX-1, and i CD68 in the glomeruli of normal mice (Bar=100 μm).
untargeted USPIOs. The high image quality, sensitivity, and resolution of MRI, combined with the limited toxicity of the targeted USPIOs [25], suggest that the anti-LOX-1 USPIOs we have described represent a potentially valuable approach for the early detection and monitoring of DN. Recent studies have demonstrated that activation of inflammatory pathways plays a central role in the early stages of DN, leading to the activation and recruitment of fibroblasts and culminating in renal fibrosis [26]. LOX-1 expression in renal tissue has been shown to play a crucial role in inflammatory changes associated with DN [27–29]. While the basal expression of LOX-1 is low, it is highly induced by its ligand, oxLDL, in addition to inflammatory cytokines, including TNF-α, interleukin1β (IL-1β), transforming growth factor-β1(TGF-β1), as well as free fatty acids, angiotensin II, endothelin-1, hyperglycemia, and hemodynamic changes [8, 30–33]. Zhang et al. have proposed that in response to activation of the LOX-1 pathway by oxidized HDL, glomerular mesangial cells adopt a dysfunctional, proinflammatory state, ultimately succumbing to apoptosis [34]. Collectively, these data afforded us a sound rationale for leveraging LOX-1 as a biomarker for the detection of early renal lesions in DN. The anticipated symptoms of hyperglycemia, dyslipidemia, polyphagia, polydipsia, and polyuria were observed in the db/db mice. Moreover, as expected, levels of urinary creatinine, urinary
protein, serum lipid, serum creatinine, and blood urea nitrogen were significantly higher in 12-week-old db/db mice than in agematched nondiabetic mice, all of which are indicative of renal dysfunction. Based on these data, 12-week-old db/db mice had pathological characteristics comparable to the early stages of human DN [3], and were therefore considered appropriate models for the current study. The PEG coating on the anti-LOX-1 USPIOs reduces plasma protein binding, retards clearance by the reticuloendothelial system, and increases particle circulation times [35], all of which increased the probability of USPIOs reaching the tissue of interest [36]. In vitro experiments showed the PEG-coated USPIO nanoparticles were not passively taken up by RAW264.7 macrophages exposed to human oxLDL for 12 h. However, Perl’s staining demonstrated that LOX-1-targeted USPIO were taken up by activated macrophages cells due to elevated membrane expression levels of LOX-1 [37]. In contrast, uptake of anti-LOX-1 USPIOs was lower in macrophages not preincubated with oxLDL due to their lower membrane expression levels of LOX-1. Collectively, these results demonstrate that LOX-1targeted PEG-coated USPIO nanoparticles selectively accumulate in activated macrophages expressing LOX-1. After in vitro analysis of USPIO uptake, we next used MR imaging to demonstrate early detection of DN by the LOX-1-targeted USPIOs in vivo. Related studies have
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reported the noninvasive detection of inflammation in a rat model of ischemic acute renal failure by USPIOenhanced MRI, with no effect on renal injury or recovery [18]. Moreover Li et al. [25] developed a novel LOX-1 targeted molecular imaging probe that bound preferentially to the plaque shoulder of the aortic arch in atherosclerotic apoE-/- mice and LDLR-/- mice, a region with extensive LOX-1 expression and macrophage accumulation. These results indicated that in vivo imaging of LOX-1 expression using an LOX-1 antibody represented a potential approach to identifying inflammatory renal lesions in early DN. In the current study, DN mice and age-matched C57BL/6 mice were examined using a T2*weighted sequence. The MR images showed a significant signal intensity decrease after 24 h administration of anti-LOX-1 USPIO in the cortex of DN mice, but not normal mice injected with targeted USPIO, nor in DN mice injected with untargeted USPIO. Moreover, Perl’s staining revealed and indicated that the iron-containing cells were mainly located in the glomerulus in DN mice injected with LOX-1-targeted USPIO particles. Furthermore, immunohistochemistry and Western blotting analysis confirmed the iron-containing cells co-localized with LOX-1/macrophage expression. In addition, we found a significant negative correlation between the percent normalized enhancement and the number of CD68-positive macrophages. In concert, these data demonstrate that LOX-1-targeted USPIOs can be used for the molecular imaging of LOX-1 enriched DN kidney lesions in vivo. Macrophages are the principle inflammatory cells in the diabetic kidney and play a crucial role in the pathogenesis of glomerulopathy lesions in DN [38], and the results indicating that the signal reduction caused by administration of LOX-1 targeted USPIOs accurately reflects the inflammatory responses characteristic of early DN. Considering the limited cell toxicity of the USPIOs in the current study compared with Gdcontaining micelles, our method represents a clinically feasible method with which for the early detection and diagnosis of DN. The limitations of this study include its undetermined relevance to humans, and the fact that the USPIO dose (10 mg/kg) exceeds that recommended for clinical applications. This dose was chosen to increase the probability of USPIO uptake by macrophages, since the half-life of these particles in rats is shorter than in humans. A final limitation of our study is the lack of information on the safety of these nanoparticles in humans, which we will evaluate in future studies. In summary, the present study demonstrates a specific, sensitive method for noninvasive imaging LOX-1 within DN renal lesions, which can potentially provide important information for the early detection of DN. Due to the limited toxicity of the USPIOs compared with Gd-containing micelles, our method represents a novel potential clinical diagnostic modality for DN.
Sources of Funding. This work was supported by the Major State Basic Research Development Program of China (973 Program) (NOs. 2013CB733800, 2013CB733803), National Natural Science Foundation of China (NOs. 81230034, 81271739, 81271637, 81401460), and Jiangsu Provincial Special Program of Medical Science (BL2013029). Conflict of Interest. The authors have declared that no competing interests exist.
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