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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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Infused MSCs persist in the lung and muscle and suppress tissue damage. (a) MSCs persisted in the lung and injured muscle 24 hours after rhabdomyolysis. RFP-labelled MSCs (red) were detected in vivo using two-photon fluorescence confocal microscopy. Nuclei were stained with DAPI (blue). Original magnification, ×600, n = 3. (b) Representative H & E-stained paraffin-embedded sections of kidney, gastrocnemius muscle and lung with or without administration of MSCs (n = 10). Mice were sacrificed 24 hours after rhabdomyolysis. (c) ELISA showed that MSC treatment significantly decreased the levels of both of the pro-inflammatory cytokines TNF-α and IL-6 and increased the anti-inflammatory cytokine IL-10. *P <0.05 versus RM + NS, n = 5. DAPI, 4,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; NS, normal saline; RM, rhabdomyolysis.
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Figure 2: Infused MSCs persist in the lung and muscle and suppress tissue damage. (a) MSCs persisted in the lung and injured muscle 24 hours after rhabdomyolysis. RFP-labelled MSCs (red) were detected in vivo using two-photon fluorescence confocal microscopy. Nuclei were stained with DAPI (blue). Original magnification, ×600, n = 3. (b) Representative H & E-stained paraffin-embedded sections of kidney, gastrocnemius muscle and lung with or without administration of MSCs (n = 10). Mice were sacrificed 24 hours after rhabdomyolysis. (c) ELISA showed that MSC treatment significantly decreased the levels of both of the pro-inflammatory cytokines TNF-α and IL-6 and increased the anti-inflammatory cytokine IL-10. *P <0.05 versus RM + NS, n = 5. DAPI, 4,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; NS, normal saline; RM, rhabdomyolysis.

Mentions: To evaluate the distribution of MSCs after AKI, RFP-labelled MSCs were infused into mice six hours after rhabdomyolysis. In vivo imaging was performed 24 hours after rhabdomyolysis using two-photon fluorescence confocal microscopy. The distribution was also confirmed by corresponding frozen sections. Twenty-four hours after rhabdomyolysis, signals were detected in the lung and gastrocnemius muscle. However, no apparent MSCs were observed in the injured kidney (Figure 2a). Rhabdomyolysis caused multiple organ damage, including significant oedema in the gastrocnemius muscles with degeneration and atrophy in muscle fibres, prominent intra-alveolar and interstitial oedema with hyaline membrane formation, as well as acute tubular necrosis. Our results showed that mice receiving MSCs displayed decreased tissue damage in the kidney, muscle and lung, compared with control mice receiving normal saline injection (Figure 2b). In addition, an ELISA showed that MSC treatment significantly decreased serum levels of the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) and increased the anti-inflammatory cytokine IL-10 (Figure 2c). These findings suggest that administration of MSCs exerted a therapeutic effect and ameliorated the progression of organ damage via unique immunomodulatory functions that inhibit the pro-inflammatory progression of injury and elicit an anti-inflammatory effect.


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)

Infused MSCs persist in the lung and muscle and suppress tissue damage. (a) MSCs persisted in the lung and injured muscle 24 hours after rhabdomyolysis. RFP-labelled MSCs (red) were detected in vivo using two-photon fluorescence confocal microscopy. Nuclei were stained with DAPI (blue). Original magnification, ×600, n = 3. (b) Representative H & E-stained paraffin-embedded sections of kidney, gastrocnemius muscle and lung with or without administration of MSCs (n = 10). Mice were sacrificed 24 hours after rhabdomyolysis. (c) ELISA showed that MSC treatment significantly decreased the levels of both of the pro-inflammatory cytokines TNF-α and IL-6 and increased the anti-inflammatory cytokine IL-10. *P <0.05 versus RM + NS, n = 5. DAPI, 4,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; NS, normal saline; RM, rhabdomyolysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Infused MSCs persist in the lung and muscle and suppress tissue damage. (a) MSCs persisted in the lung and injured muscle 24 hours after rhabdomyolysis. RFP-labelled MSCs (red) were detected in vivo using two-photon fluorescence confocal microscopy. Nuclei were stained with DAPI (blue). Original magnification, ×600, n = 3. (b) Representative H & E-stained paraffin-embedded sections of kidney, gastrocnemius muscle and lung with or without administration of MSCs (n = 10). Mice were sacrificed 24 hours after rhabdomyolysis. (c) ELISA showed that MSC treatment significantly decreased the levels of both of the pro-inflammatory cytokines TNF-α and IL-6 and increased the anti-inflammatory cytokine IL-10. *P <0.05 versus RM + NS, n = 5. DAPI, 4,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; NS, normal saline; RM, rhabdomyolysis.
Mentions: To evaluate the distribution of MSCs after AKI, RFP-labelled MSCs were infused into mice six hours after rhabdomyolysis. In vivo imaging was performed 24 hours after rhabdomyolysis using two-photon fluorescence confocal microscopy. The distribution was also confirmed by corresponding frozen sections. Twenty-four hours after rhabdomyolysis, signals were detected in the lung and gastrocnemius muscle. However, no apparent MSCs were observed in the injured kidney (Figure 2a). Rhabdomyolysis caused multiple organ damage, including significant oedema in the gastrocnemius muscles with degeneration and atrophy in muscle fibres, prominent intra-alveolar and interstitial oedema with hyaline membrane formation, as well as acute tubular necrosis. Our results showed that mice receiving MSCs displayed decreased tissue damage in the kidney, muscle and lung, compared with control mice receiving normal saline injection (Figure 2b). In addition, an ELISA showed that MSC treatment significantly decreased serum levels of the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) and increased the anti-inflammatory cytokine IL-10 (Figure 2c). These findings suggest that administration of MSCs exerted a therapeutic effect and ameliorated the progression of organ damage via unique immunomodulatory functions that inhibit the pro-inflammatory progression of injury and elicit an anti-inflammatory effect.

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