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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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Comparison of cell surface markers and inflammatory factor expression in M0, M1 and M2 macrophages. (a) Cell surface marker staining of F4/80 and CD206 expression in M0, M1 and M2 macrophages. RAW264.7 macrophages cultured in normal medium were defined as M0 with significant F4/80 and negative CD206 expression. RAW264.7 macrophages stimulated with LPS were defined as M1 with significant F4/80 and weak CD206 expression. RAW264.7 macrophages stimulated with LPS and co-cultured with MSCs in transwells were defined as M2 with significant expression of F4/80 and CD206. (b) ELISA showed that MSC treatment significantly decreased the levels of the pro-inflammatory cytokines TNF-α and IL-6 and increased the levels of the anti-inflammatory cytokine IL-10 in culture medium. *P <0.05 versus M0, #P <0.05 versus M2, n = 3. (c-d) MSCs increased the percentage of CD206+ IL-10+ cells in RAW264.7 macrophages after co-culture for 72 hours. #P <0.05 versus M0 or M1, n = 3. MSCs, mesenchymal stem cells.
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Figure 7: Comparison of cell surface markers and inflammatory factor expression in M0, M1 and M2 macrophages. (a) Cell surface marker staining of F4/80 and CD206 expression in M0, M1 and M2 macrophages. RAW264.7 macrophages cultured in normal medium were defined as M0 with significant F4/80 and negative CD206 expression. RAW264.7 macrophages stimulated with LPS were defined as M1 with significant F4/80 and weak CD206 expression. RAW264.7 macrophages stimulated with LPS and co-cultured with MSCs in transwells were defined as M2 with significant expression of F4/80 and CD206. (b) ELISA showed that MSC treatment significantly decreased the levels of the pro-inflammatory cytokines TNF-α and IL-6 and increased the levels of the anti-inflammatory cytokine IL-10 in culture medium. *P <0.05 versus M0, #P <0.05 versus M2, n = 3. (c-d) MSCs increased the percentage of CD206+ IL-10+ cells in RAW264.7 macrophages after co-culture for 72 hours. #P <0.05 versus M0 or M1, n = 3. MSCs, mesenchymal stem cells.

Mentions: Next, we investigated the hypothesis that the interaction of MSCs with macrophages plays a significant role in their anti-inflammatory and immune modulatory effects. RAW 264.7 cells, a murine macrophage-like cell line, were cultured in normal medium and defined as M0, and M0 stimulated with LPS was defined as M1. M1 co-cultured with MSCs for 72 hours with significantly increased expression of CD206, as assessed using polarised immunofluorescence staining, was defined as M2 (Figure 7a). To determine whether secreted factors were responsible for the transition of the macrophage phenotype, transwell inserts were used during co-culture to prevent direct physical contact between the MSCs and macrophages. As MSC-co-cultured macrophages expressed the anti-inflammatory macrophage marker CD206, intracellular cytokine staining utilising established stimulation protocols [25,26] was performed to further characterise their immunophenotype. Flow cytometry analysis showed that MSC-co-cultured M2 macrophages produced more IL-10 (Figure 7c and d) compared to M0 and M1 macrophages. In addition, ELISA analysis showed that after co-culture with MSCs, the levels of the pro-inflammatory cytokines TNF-a and IL-6 in the medium of RAW 264.7 cells were significantly decreased, while that of the anti-inflammatory cytokine IL-10 was accordingly increased (Figure 7b).


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)

Comparison of cell surface markers and inflammatory factor expression in M0, M1 and M2 macrophages. (a) Cell surface marker staining of F4/80 and CD206 expression in M0, M1 and M2 macrophages. RAW264.7 macrophages cultured in normal medium were defined as M0 with significant F4/80 and negative CD206 expression. RAW264.7 macrophages stimulated with LPS were defined as M1 with significant F4/80 and weak CD206 expression. RAW264.7 macrophages stimulated with LPS and co-cultured with MSCs in transwells were defined as M2 with significant expression of F4/80 and CD206. (b) ELISA showed that MSC treatment significantly decreased the levels of the pro-inflammatory cytokines TNF-α and IL-6 and increased the levels of the anti-inflammatory cytokine IL-10 in culture medium. *P <0.05 versus M0, #P <0.05 versus M2, n = 3. (c-d) MSCs increased the percentage of CD206+ IL-10+ cells in RAW264.7 macrophages after co-culture for 72 hours. #P <0.05 versus M0 or M1, n = 3. MSCs, mesenchymal stem cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Comparison of cell surface markers and inflammatory factor expression in M0, M1 and M2 macrophages. (a) Cell surface marker staining of F4/80 and CD206 expression in M0, M1 and M2 macrophages. RAW264.7 macrophages cultured in normal medium were defined as M0 with significant F4/80 and negative CD206 expression. RAW264.7 macrophages stimulated with LPS were defined as M1 with significant F4/80 and weak CD206 expression. RAW264.7 macrophages stimulated with LPS and co-cultured with MSCs in transwells were defined as M2 with significant expression of F4/80 and CD206. (b) ELISA showed that MSC treatment significantly decreased the levels of the pro-inflammatory cytokines TNF-α and IL-6 and increased the levels of the anti-inflammatory cytokine IL-10 in culture medium. *P <0.05 versus M0, #P <0.05 versus M2, n = 3. (c-d) MSCs increased the percentage of CD206+ IL-10+ cells in RAW264.7 macrophages after co-culture for 72 hours. #P <0.05 versus M0 or M1, n = 3. MSCs, mesenchymal stem cells.
Mentions: Next, we investigated the hypothesis that the interaction of MSCs with macrophages plays a significant role in their anti-inflammatory and immune modulatory effects. RAW 264.7 cells, a murine macrophage-like cell line, were cultured in normal medium and defined as M0, and M0 stimulated with LPS was defined as M1. M1 co-cultured with MSCs for 72 hours with significantly increased expression of CD206, as assessed using polarised immunofluorescence staining, was defined as M2 (Figure 7a). To determine whether secreted factors were responsible for the transition of the macrophage phenotype, transwell inserts were used during co-culture to prevent direct physical contact between the MSCs and macrophages. As MSC-co-cultured macrophages expressed the anti-inflammatory macrophage marker CD206, intracellular cytokine staining utilising established stimulation protocols [25,26] was performed to further characterise their immunophenotype. Flow cytometry analysis showed that MSC-co-cultured M2 macrophages produced more IL-10 (Figure 7c and d) compared to M0 and M1 macrophages. In addition, ELISA analysis showed that after co-culture with MSCs, the levels of the pro-inflammatory cytokines TNF-a and IL-6 in the medium of RAW 264.7 cells were significantly decreased, while that of the anti-inflammatory cytokine IL-10 was accordingly increased (Figure 7b).

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