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Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages.

Geng Y, Zhang L, Fu B, Zhang J, Hong Q, Hu J, Li D, Luo C, Cui S, Zhu F, Chen X - Stem Cell Res Ther (2014)

Bottom Line: This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI.MSCs were injected into glycerol-induced rhabdomyolysis mice.The concentrations of IL-10, IL-6 and tumor necrosis factor α were evaluated using enzyme-linked immunosorbent assay.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI.

Methods: MSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate.

Results: In vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL-6 and tumor necrosis factor α were evaluated using enzyme-linked immunosorbent assay. Furthermore, macrophage-depleted mice with intramuscular injection of glycerol were subjected to a single injection of different types of RAW 264.7 macrophages. Mice infused with M0 and M1 macrophages suffered a more severe histological and functional injury, while mice transfused with MSC-educated M2 macrophages showed reduced kidney injury.

Conclusions: Our findings suggested that MSCs can ameliorate rhabdomyolysis-induced AKI via the activation of macrophages to a trophic M2 phenotype, which supports the transition from tubule injury to tubule repair.

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Related in: MedlinePlus

Experimental schedule of macrophage depletion 24 hours after rhabdomyolysis. A bolus of 100 μL/10 g body weight of liposomal-encapsulated clodronate (LC) was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a liposomal vehicle (LV) was administered as a control. AKI, acute kidney injury; MSC, mesenchymal stem cells.
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Figure 5: Experimental schedule of macrophage depletion 24 hours after rhabdomyolysis. A bolus of 100 μL/10 g body weight of liposomal-encapsulated clodronate (LC) was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a liposomal vehicle (LV) was administered as a control. AKI, acute kidney injury; MSC, mesenchymal stem cells.

Mentions: It was determined that CD206+, non-inflammatory (M2) macrophages predominate in the RM + MSCs group. Twenty-four hours after rhabdomyolysis, western blotting analysis revealed that RM mice treated with MSCs showed strong expression of CD206 in the kidney, while RM mice infused with normal saline showed weak expression. Independent of receiving normal saline or MSC treatment, the sham control did not show any CD206 expression (Figure 4a). In addition, at 24 hours, a bolus of 100 μL/10 g body weight of LC was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a LV was administered as a control (Figure 5). Renal histopathology and function analyses showed that depletion of M2-polarised macrophages can resume tubular injury at 72 hours (Figure 4b, 4c). In MSC-treated mice, animals injected with LC compared with LV, still showed higher levels of SCr and BUN and aggravation of tubular injury 72 hours after rhabdomyolysis.


Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages.

Geng Y, Zhang L, Fu B, Zhang J, Hong Q, Hu J, Li D, Luo C, Cui S, Zhu F, Chen X - Stem Cell Res Ther (2014)

Experimental schedule of macrophage depletion 24 hours after rhabdomyolysis. A bolus of 100 μL/10 g body weight of liposomal-encapsulated clodronate (LC) was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a liposomal vehicle (LV) was administered as a control. AKI, acute kidney injury; MSC, mesenchymal stem cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230233&req=5

Figure 5: Experimental schedule of macrophage depletion 24 hours after rhabdomyolysis. A bolus of 100 μL/10 g body weight of liposomal-encapsulated clodronate (LC) was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a liposomal vehicle (LV) was administered as a control. AKI, acute kidney injury; MSC, mesenchymal stem cells.
Mentions: It was determined that CD206+, non-inflammatory (M2) macrophages predominate in the RM + MSCs group. Twenty-four hours after rhabdomyolysis, western blotting analysis revealed that RM mice treated with MSCs showed strong expression of CD206 in the kidney, while RM mice infused with normal saline showed weak expression. Independent of receiving normal saline or MSC treatment, the sham control did not show any CD206 expression (Figure 4a). In addition, at 24 hours, a bolus of 100 μL/10 g body weight of LC was injected into MSC-treated AKI mice to deplete M2-polarised macrophages, and a LV was administered as a control (Figure 5). Renal histopathology and function analyses showed that depletion of M2-polarised macrophages can resume tubular injury at 72 hours (Figure 4b, 4c). In MSC-treated mice, animals injected with LC compared with LV, still showed higher levels of SCr and BUN and aggravation of tubular injury 72 hours after rhabdomyolysis.

Bottom Line: This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI.MSCs were injected into glycerol-induced rhabdomyolysis mice.The concentrations of IL-10, IL-6 and tumor necrosis factor α were evaluated using enzyme-linked immunosorbent assay.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI.

Methods: MSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate.

Results: In vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL-6 and tumor necrosis factor α were evaluated using enzyme-linked immunosorbent assay. Furthermore, macrophage-depleted mice with intramuscular injection of glycerol were subjected to a single injection of different types of RAW 264.7 macrophages. Mice infused with M0 and M1 macrophages suffered a more severe histological and functional injury, while mice transfused with MSC-educated M2 macrophages showed reduced kidney injury.

Conclusions: Our findings suggested that MSCs can ameliorate rhabdomyolysis-induced AKI via the activation of macrophages to a trophic M2 phenotype, which supports the transition from tubule injury to tubule repair.

Show MeSH
Related in: MedlinePlus