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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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MSC–educated M2 macrophages improve kidney injury and promote kidney repair. Mice treated with LV or LC were subjected to rhabdomyolysis followed by infusion of 1 × 107 M0, M1 or M2 macrophages immediately after glycerol injection. (a-c) Serum creatinine (SCr) values, blood urea nitrogen (BUN) values and acute tubular necrosis (ATN) scores are shown for mice 72 hours after rhabdomyolysis. #P <0.05 versus rhabdomyolysis LV alone, M0 or M1 groups, n = 5. (d) Representative renal histopathology of different groups is shown 72 hours after rhabdomyolysis. LC, liposomal clodronate; LV, liposomal vehicle; MSC, mesenchymal stem cell.
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Figure 9: MSC–educated M2 macrophages improve kidney injury and promote kidney repair. Mice treated with LV or LC were subjected to rhabdomyolysis followed by infusion of 1 × 107 M0, M1 or M2 macrophages immediately after glycerol injection. (a-c) Serum creatinine (SCr) values, blood urea nitrogen (BUN) values and acute tubular necrosis (ATN) scores are shown for mice 72 hours after rhabdomyolysis. #P <0.05 versus rhabdomyolysis LV alone, M0 or M1 groups, n = 5. (d) Representative renal histopathology of different groups is shown 72 hours after rhabdomyolysis. LC, liposomal clodronate; LV, liposomal vehicle; MSC, mesenchymal stem cell.

Mentions: To determine whether MSC-induced macrophages exert different effects following kidney injury, circulating and tissue myeloid phagocytes, including monocytes and resident macrophages, were depleted by LC. Mice with LV infusion were treated as a negative control. After two days, all mice received an intramuscular injection of glycerol to induce rhabdomyolysis. Next, 1 × 107 pre-treated macrophages were injected into macrophage-depleted mice (Figure 8). Animals infused with M0 macrophages or M1 macrophages demonstrated increased levels of SCr and BUN that were indistinguishable from LV-treated control animals 72 hours after injury. Simultaneously, M2 macrophages also ameliorated rhabdomyolysis-induced renal injury (Figure 9a, 9b). Examination of renal histology (Figure 9d) and tubular injury scoring (Figure 9c) 72 hours after rhabdomyolysis confirmed that tubule damage was less severe in M2 macrophage-treated animals compared with LV alone or M0 and M1 macrophage-infused mice. Thus, these data demonstrated that MSCs ameliorate rhabdomyolysis-induced AKI via eliciting the polarisation of M2 macrophages.


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)

MSC–educated M2 macrophages improve kidney injury and promote kidney repair. Mice treated with LV or LC were subjected to rhabdomyolysis followed by infusion of 1 × 107 M0, M1 or M2 macrophages immediately after glycerol injection. (a-c) Serum creatinine (SCr) values, blood urea nitrogen (BUN) values and acute tubular necrosis (ATN) scores are shown for mice 72 hours after rhabdomyolysis. #P <0.05 versus rhabdomyolysis LV alone, M0 or M1 groups, n = 5. (d) Representative renal histopathology of different groups is shown 72 hours after rhabdomyolysis. LC, liposomal clodronate; LV, liposomal vehicle; MSC, mesenchymal stem cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: MSC–educated M2 macrophages improve kidney injury and promote kidney repair. Mice treated with LV or LC were subjected to rhabdomyolysis followed by infusion of 1 × 107 M0, M1 or M2 macrophages immediately after glycerol injection. (a-c) Serum creatinine (SCr) values, blood urea nitrogen (BUN) values and acute tubular necrosis (ATN) scores are shown for mice 72 hours after rhabdomyolysis. #P <0.05 versus rhabdomyolysis LV alone, M0 or M1 groups, n = 5. (d) Representative renal histopathology of different groups is shown 72 hours after rhabdomyolysis. LC, liposomal clodronate; LV, liposomal vehicle; MSC, mesenchymal stem cell.
Mentions: To determine whether MSC-induced macrophages exert different effects following kidney injury, circulating and tissue myeloid phagocytes, including monocytes and resident macrophages, were depleted by LC. Mice with LV infusion were treated as a negative control. After two days, all mice received an intramuscular injection of glycerol to induce rhabdomyolysis. Next, 1 × 107 pre-treated macrophages were injected into macrophage-depleted mice (Figure 8). Animals infused with M0 macrophages or M1 macrophages demonstrated increased levels of SCr and BUN that were indistinguishable from LV-treated control animals 72 hours after injury. Simultaneously, M2 macrophages also ameliorated rhabdomyolysis-induced renal injury (Figure 9a, 9b). Examination of renal histology (Figure 9d) and tubular injury scoring (Figure 9c) 72 hours after rhabdomyolysis confirmed that tubule damage was less severe in M2 macrophage-treated animals compared with LV alone or M0 and M1 macrophage-infused mice. Thus, these data demonstrated that MSCs ameliorate rhabdomyolysis-induced AKI via eliciting the polarisation of M2 macrophages.

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