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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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Effect of co-culture with MSCs on mRNA expression. MSCs reduce macrophage gene transcriptional activity of TNF-a and iNOS and promote gene transcriptional activity of IL-10 and MR in the RAW264.7 cell line and macrophages of the C57BL/6 strain. #P <0.01 versus M0 and M1. iNOS, inducible nitric oxide synthase; MSCs, mesenchymal stem cells.
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Figure 6: Effect of co-culture with MSCs on mRNA expression. MSCs reduce macrophage gene transcriptional activity of TNF-a and iNOS and promote gene transcriptional activity of IL-10 and MR in the RAW264.7 cell line and macrophages of the C57BL/6 strain. #P <0.01 versus M0 and M1. iNOS, inducible nitric oxide synthase; MSCs, mesenchymal stem cells.

Mentions: To investigate whether RAW264.7 cells have similar immunogenicity as macrophages of the C57BL/6 strain, we examined the level of TNF-α, iNOS, IL-10 and MR using RT-PCR. Our results showed that compared with the M0 and M1 groups, the mRNA expression levels of TNF-α and iNOS were significantly decreased and the mRNA expression levels of IL-10 and MR were significantly increased in co-culture groups of RAW264.7 cells and macrophages of the C57BL/6 strain at 24 hours (Figure 6).


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)

Effect of co-culture with MSCs on mRNA expression. MSCs reduce macrophage gene transcriptional activity of TNF-a and iNOS and promote gene transcriptional activity of IL-10 and MR in the RAW264.7 cell line and macrophages of the C57BL/6 strain. #P <0.01 versus M0 and M1. iNOS, inducible nitric oxide synthase; MSCs, 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 6: Effect of co-culture with MSCs on mRNA expression. MSCs reduce macrophage gene transcriptional activity of TNF-a and iNOS and promote gene transcriptional activity of IL-10 and MR in the RAW264.7 cell line and macrophages of the C57BL/6 strain. #P <0.01 versus M0 and M1. iNOS, inducible nitric oxide synthase; MSCs, mesenchymal stem cells.
Mentions: To investigate whether RAW264.7 cells have similar immunogenicity as macrophages of the C57BL/6 strain, we examined the level of TNF-α, iNOS, IL-10 and MR using RT-PCR. Our results showed that compared with the M0 and M1 groups, the mRNA expression levels of TNF-α and iNOS were significantly decreased and the mRNA expression levels of IL-10 and MR were significantly increased in co-culture groups of RAW264.7 cells and macrophages of the C57BL/6 strain at 24 hours (Figure 6).

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