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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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MSCs increase infiltration of M2-polarised macrophages in rhabdomyolysis kidney and promote tubular cell regeneration. (a) Representative confocal microscopy images of macrophages (F4/80) and M2-polarised macrophage (CD206) infiltration in kidneys after saline or MSC infusion. (b) PCNA–positive nuclei within the tubuli of glycerol-treated mice treated with saline or MSCs at various time intervals (24, 48 and 72 hours). (c) Representative western blotting analysis showing time-dependent increases in mannose receptor (CD206) expression induced by MSC treatment. *P <0.05 versus RM + NS, n = 3. MSC, mesenchymal stem cell; NS, normal saline; PCNA, proliferating cell nuclear antigen; RM, rhabdomyolysis.
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Figure 3: MSCs increase infiltration of M2-polarised macrophages in rhabdomyolysis kidney and promote tubular cell regeneration. (a) Representative confocal microscopy images of macrophages (F4/80) and M2-polarised macrophage (CD206) infiltration in kidneys after saline or MSC infusion. (b) PCNA–positive nuclei within the tubuli of glycerol-treated mice treated with saline or MSCs at various time intervals (24, 48 and 72 hours). (c) Representative western blotting analysis showing time-dependent increases in mannose receptor (CD206) expression induced by MSC treatment. *P <0.05 versus RM + NS, n = 3. MSC, mesenchymal stem cell; NS, normal saline; PCNA, proliferating cell nuclear antigen; RM, rhabdomyolysis.

Mentions: Representative confocal micrographs of kidneys showed the presence of macrophages and M2-polarised macrophages in RM + NS and RM + MSCs groups after 24, 48 and 72 hours of rhabdomyolysis (Figure 3a). Macrophages were detected using green fluorescence with an antibody specific for the mouse macrophage marker F4/80, and M2-polarised macrophages were detected using red fluorescence with an antibody specific for the CD206 marker. Nuclei were counterstained with DAPI (blue). Macrophage infiltration increased after rhabdomyolysis, and treatment with MSCs accelerated M2-polarised macrophage infiltration, as represented by immunostaining. Western blotting analysis demonstrated a time-dependent increase in CD206 protein expression in the kidney after rhabdomyolysis (Figure 3c). This observation suggested that macrophage presentation in MSC treatment had adopted a phenotype similar to that of alternatively-activated macrophages. As shown in Figure 3b, MSCs also significantly enhanced tubular cell proliferation compared with saline treatment alone, as detected by PCNA-positive cells after 24, 48 and 72 hours of rhabdomyolysis. These compelling findings suggested that MSCs were capable of promoting infiltration of alternatively-activated macrophages in the wounded kidney, which potentially contributed to the regulation of the inflammatory response and enhanced the healing of 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)

MSCs increase infiltration of M2-polarised macrophages in rhabdomyolysis kidney and promote tubular cell regeneration. (a) Representative confocal microscopy images of macrophages (F4/80) and M2-polarised macrophage (CD206) infiltration in kidneys after saline or MSC infusion. (b) PCNA–positive nuclei within the tubuli of glycerol-treated mice treated with saline or MSCs at various time intervals (24, 48 and 72 hours). (c) Representative western blotting analysis showing time-dependent increases in mannose receptor (CD206) expression induced by MSC treatment. *P <0.05 versus RM + NS, n = 3. MSC, mesenchymal stem cell; NS, normal saline; PCNA, proliferating cell nuclear antigen; RM, rhabdomyolysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: MSCs increase infiltration of M2-polarised macrophages in rhabdomyolysis kidney and promote tubular cell regeneration. (a) Representative confocal microscopy images of macrophages (F4/80) and M2-polarised macrophage (CD206) infiltration in kidneys after saline or MSC infusion. (b) PCNA–positive nuclei within the tubuli of glycerol-treated mice treated with saline or MSCs at various time intervals (24, 48 and 72 hours). (c) Representative western blotting analysis showing time-dependent increases in mannose receptor (CD206) expression induced by MSC treatment. *P <0.05 versus RM + NS, n = 3. MSC, mesenchymal stem cell; NS, normal saline; PCNA, proliferating cell nuclear antigen; RM, rhabdomyolysis.
Mentions: Representative confocal micrographs of kidneys showed the presence of macrophages and M2-polarised macrophages in RM + NS and RM + MSCs groups after 24, 48 and 72 hours of rhabdomyolysis (Figure 3a). Macrophages were detected using green fluorescence with an antibody specific for the mouse macrophage marker F4/80, and M2-polarised macrophages were detected using red fluorescence with an antibody specific for the CD206 marker. Nuclei were counterstained with DAPI (blue). Macrophage infiltration increased after rhabdomyolysis, and treatment with MSCs accelerated M2-polarised macrophage infiltration, as represented by immunostaining. Western blotting analysis demonstrated a time-dependent increase in CD206 protein expression in the kidney after rhabdomyolysis (Figure 3c). This observation suggested that macrophage presentation in MSC treatment had adopted a phenotype similar to that of alternatively-activated macrophages. As shown in Figure 3b, MSCs also significantly enhanced tubular cell proliferation compared with saline treatment alone, as detected by PCNA-positive cells after 24, 48 and 72 hours of rhabdomyolysis. These compelling findings suggested that MSCs were capable of promoting infiltration of alternatively-activated macrophages in the wounded kidney, which potentially contributed to the regulation of the inflammatory response and enhanced the healing of 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