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. 2016 Apr 21;6(6):896-914.
doi: 10.7150/thno.14915. eCollection 2016.

CD163-Macrophages Are Involved in Rhabdomyolysis-Induced Kidney Injury and May Be Detected by MRI with Targeted Gold-Coated Iron Oxide Nanoparticles

Alfonso Rubio-Navarro  1 , Mónica Carril  2 , Daniel Padro  3 , Melanie Guerrero-Hue  1 , Carlos Tarín  1 , Rafael Samaniego  4 , Pablo Cannata  5 , Ainhoa Cano  3 , Juan Manuel Amaro Villalobos  1 , Ángel Manuel Sevillano  6 , Claudia Yuste  6 , Eduardo Gutiérrez  6 , Manuel Praga  6 , Jesús Egido  7 , Juan Antonio Moreno  1
Affiliations

CD163-Macrophages Are Involved in Rhabdomyolysis-Induced Kidney Injury and May Be Detected by MRI with Targeted Gold-Coated Iron Oxide Nanoparticles

Alfonso Rubio-Navarro et al. Theranostics. .

Abstract

Macrophages play an important role in rhabdomyolysis-acute kidney injury (AKI), although the molecular mechanisms involved in macrophage differentiation are poorly understood. We analyzed the expression and regulation of CD163, a membrane receptor mainly expressed by anti-inflammatory M2 macrophages, in rhabdomyolysis-AKI and developed targeted probes for its specific detection in vivo by MRI. Intramuscular injection of glycerol in mice promoted an early inflammatory response, with elevated proportion of M1 macrophages, and partial differentiation towards a M2 phenotype in later stages, where increased CD163 expression was observed. Immunohistological studies confirmed the presence of CD163-macrophages in human rhabdomyolysis-AKI. In cultured macrophages, myoglobin upregulated CD163 expression via HO-1/IL-10 axis. Moreover, we developed gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody that specifically targeted CD163 in kidneys from glycerol-injected mice, as determined by MRI studies, and confirmed by electron microscopy and immunological analysis. Our findings are the first to demonstrate that CD163 is present in both human and experimental rhabdomyolysis-induced AKI, suggesting an important role of this molecule in this pathological condition. Therefore, the use of probes targeting CD163-macrophages by MRI may provide important information about the cellular composition of renal lesion in rhabdomyolysis.

Keywords: CD163; MRI; acute kidney injury.; gold coated iron oxide nanoparticles; macrophages; rhabdomyolysis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Rhabdomyolysis promotes renal damage by oxidative stress and tubular apoptosis. C57BI/6 mice (males, 12 weeks old) were injected with saline or 10 ml/kg 50% glycerol in each thigh caudal muscle. Creatinine (A), BUN (B) and urinary myoglobin (C) were elevated from day 1 to 3, decreasing at day 7. (D) Representative images showing hematoxylin and eosin staining, confocal TUNEL images, and immunhistochemical 4-HNE, HO-1 and Ferritin images of mouse kidneys, scale bar 50 µM. (E) Quantitative TUNEL positive cells analysis in mice with rhabdomyolysis. Expression of tubular injury biomarkers Kim-1 (F) and Ngal (G), as determined by real time RT-PCR, in kidneys from mice with rhabdomyolysis. HO-1 expression, as determined by western-blot (H) and RT-PCR (I) was elevated 24h after glycerol-injection; whereas ferritin presence was increased from day 1 to day 7, as determined by Western-blot (J). Mice (n=5) per day-group. Results are expressed as mean ± SE. * p<0.05 vs non-treated mice.
Figure 2
Figure 2
Rhabdomyolysis induces macrophages recruitment in murine kidneys. (A) Expression of pro-inflammatory (CCL2, CCL5, IFN-γ, TNF-α) and anti-inflammatory (IL-10 and IL-4) molecules, as determined by real time RT-PCR, in mice with rhabdomyolysis (B) Representative inmunohistochemistry (left panel) and semiquantitative assessment (right panel) of total infiltrating macrophages (F4/80) and CD163-macrophages in kidneys from mice with rhabdomyolysis, scale bar 50 µM. Expression of M1 (Arg2) and M2 (Arg1, CD163 and mannose receptor (MR)) macrophage markers, as determined by RT-PCR (C) and western-blot (D). Arg2 and Arg1 protein expression values were corrected by loading control (Tubulin) and expressed as M2/M1 ratio (E). Mice (n=5) per day-group. Results are expressed as mean ± SE. * p<0.05 vs non-treated mice. (F) Representative serial micrographs of CD206 and CD163, (white arrows indicate the presence of CD206 in CD163-macrophages), scale bar 50 µM.
Figure 3
Figure 3
Rhabdomyolysis induces renal macrophages recruitment in a patient with rhabdomyolysis-induced AKI. (A) Creatinine (Cr) and creatine kinase (CK) serum levels indicated severe rhabdomyolysis-AKI in a 73-year-old patient that required dialysis for 14 days. Renal biopsy was performed on day 24. (B) Representative inmunohistochemistry showing hematoxylin and eosin staining (upper panel), total infiltrating macrophages (CD68, middle panel) and CD163-macrophages (CD163, lower panel) in the renal biopsy from with rhabdomyolysis-associated AKI, scale bar 50 µM. Healthy renal tissue was used as control. Histological analysis reported increased interstitial macrophage infiltration and the presence of acute tubular damage (white arrows), with tubules showing dilatation and flattening of the epithelial cells and containing brown granular material and protein casts (grey arrows), heme-iron deposits (yellow arrow). The rectangle shows the region of interest for which high-magnification images are shown in the right panels. (C) Representative confocal microscopy images showing co-localization of CD68 (green) and CD163 (red) in macrophages within kidney of the patient with rhabdomyolysis, scale bar 10 µM. Nuclei were stained with DAPI (blue). TL: tubular lumen. (D) Representative confocal microscopy images (left panel) and semiquantitative assessment (right panel), showing co-localization of CD206 (green), CD163L1 (green) and CD163 (red) in macrophages within kidney of the patient with rhabdomyolysis, scale bar 10 µM. Nuclei were stained with DAPI (blue).
Figure 4
Figure 4
Myoglobin induces CD163 expression in mouse peritoneal macrophages through HO-1 and IL-10 induction. Macrophages were isolated from the mouse peritoneal cavity and treated with myoglobin (Mb) (0-2.5 mg/mL) for 48h. Dose- (A) and time-(B) enhanced mRNA expression of IL-10 and HO-1, as determined by real time RT-PCR, in myoglobin-treated macrophages. (C) Representative confocal microscopy images showing enhanced HO-1 expression in macrophages treated with myoglobin (1mg/mL) for 24h. Scale bar 50 µM (D) IL-10 release was determined by ELISA in cell supernatants. (E-F) Expression of CD163 in macrophages treated with myoglobin (1mg/mL) or equimolar concentration of heme (60µM) for 48h in presence or absence of the HO-1 inducer CoPP or an IL-10 blocking antibody (1µg/mL), as determined by RT-PCR. (G) Expression of M1 (CCL2, Arg2, TNF-α, IL-12) and M2 markers (Arg1) in macrophages stimulated with myoglobin (1mg/mL), as determined by RT-PCR. Results are expressed as mean±SE of at least three independent experiments. * p<0.05 as compared with non-treated cells, ≠ p< 0.05 as compared with cells stimulated with Mb, # p< 0.05 as compared with cells treated with Mb for 6 hours.
Figure 5
Figure 5
In vivo detection of CD163 by MRI in the glycerol model of rhabdomyolysis (A) Schematic representation of the nanoparticles administered in mice. Nanoparticles consisted of a gold-coated iron oxide core covered with thiol ligands bearing mannose and a carboxylic acid. ProtG was covalently linked through a peptide bond to the carboxylic moieties and anti-CD163 (NP-CD163) or IgG antibodies (NP-IgG) were subsequently grafted on them. (B) TEM micrograph of NP-CD163 and (C) UV-Vis spectra of gold-coated NPs before and after IgG antibodies conjugation. (D) Graph showing the normalized T2 values obtained from MRI images of mouse peritoneal macrophages incubated with NP-CD163 or NP-IgG (as control) in presence of dexamethasone for 24h (DXM, a CD163 inducer). Mean±SD of 3 independent experiments. * p<0.05 vs. NP-CD163 without dexamethasone. The corresponding MRI phantoms are shown below the graph. (E) Representative magnetic resonance images obtained pre (0h) and post (48h) nanoparticles injection in mice with rhabdomyolysis. Graph showing the contrast-to-noise-ratio (CNR) of the kidney cortex with respect to muscle pre- and post-nanoparticle injection (F) and normalized-enhancement-ratio (NER) of the kidney cortex with respect to muscle 48 hours after nanoparticle injection (G) in mice with rhabdomyolysis and control. * p<0.05 vs pre-nanoparticle injection. † p<0.05 vs healthy mice or mice with rhabdomyolysis and injected with NP-IgG. (H) Detection of nanoparticles in mice by using TEM. Left panels shows 4000x magnification of renal cortex. White arrows show the presence of infiltrating macrophages in the kidney of mice 5 days after i.m. injection of glycerol. Central panel shows 12000 x magnification of macrophages. The rectangle shows the region of interest for which high-magnification images are shown in the right panels (120000 x magnification). Black arrows show the presence of nanoparticles in mice with rhabdomyolysis and injected with NP-CD163, but not in those mice treated with NP-IgG.
Figure 6
Figure 6
Validation of the presence of CD163 in kidneys harvested after imaging studies. (A) Representative images showing hematoxylin and eosin, total macrophages (F4/80) and CD163-macrophages in kidneys obtained from mice after imaging studies, scale bar 50 µM. (B) Representative serial micrographs of F4/80 and CD163 (white arrows indicate the presence of CD163 in CD68-macrophages), scale bar 50 µM. (C) Semiquantitative assessment of total infiltrating macrophages (F4/80) and CD163-positive staining and CD163 gene expression, as determined by RT-PCR (D). Expression of M1 (Arg2) and M2 (Arg1) macrophage markers, as determined by RT-PCR (E) and western-blot (F-G). Mice (n=5) per group. * p<0.05 vs non-treated mice.
Figure 7
Figure 7
Rhabdomyolysis induces renal fibrosis. Representative images showing kidney collagen content by Sirius red (A) and semiquantitative assessment (B) in mice with rhabdomyolysis, scale bar 100 µM. Expression of fibronectin (FN), type I collagen (Col I), alpha smooth muscle actin (α-SMA) and the pro-fibrotic mediator transforming growth factor (TGF-β), as determined by RT-PCR (C) and western-blot (D). Protein expression values were corrected by loading control (Tubulin). Mice (n=5) per group. * p<0.05 vs non-treated mice. In other hand, to determine whether macrophages were involved in rhabdomyolysis-associated fibrosis, we analyzed the expression of fibronectin (FN), type I collagen (Col I) platelet derived growth factor (PDGF), connective tissue growth factor (CTGF) and transforming growth factor type beta (TGF-β ) at mRNA level (F, H) or protein secretion (G) in supernatants from myoglobin (1mg/mL)-stimulated murine peritoneal macrophages. Murine tubular epithelial cell line (MCT) were also treated with myoglobin (1mg/mL) to determine the expression (I) and release (J) of extracellular matrix proteins (Col I and FN). Results are expressed as mean±SE of at least three independent experiments. * p<0.05 as compared with non-treated cells.
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