Limits...
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.

Show MeSH

Related in: MedlinePlus

Mesenchymal stem cells (MSCs) ameliorate rhabdomyolysis (RM)-induced acute kidney injury (AKI). (a-c) Compared with animals treated with saline, infused MSCs significantly reduced serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)) and serum phosphocreatine kinase (CK(c)) levels 24, 48 and 72 hours after RM, and there were no significant changes following treatment of sham mice. *P <0.05 versus sham, #P <0.05 versus rhabdomyolysis treated with saline at the corresponding times (n = 10). (d) MSC therapy markedly reduced acute tubular necrosis (ATN) scores 72 hours after rhabdomyolysis. (e-h) Treatment with the sham-operated control (Sham + NS and Sham + MSCs) had no effect on renal histopathological parameters (e and f), while MSC therapy markedly improved tubular injury (h), and mice treated with saline showed severe tubular injury (g) (HPFs, ×400, n = 10). HPFs, high-power fields; NS, normal saline.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Mesenchymal stem cells (MSCs) ameliorate rhabdomyolysis (RM)-induced acute kidney injury (AKI). (a-c) Compared with animals treated with saline, infused MSCs significantly reduced serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)) and serum phosphocreatine kinase (CK(c)) levels 24, 48 and 72 hours after RM, and there were no significant changes following treatment of sham mice. *P <0.05 versus sham, #P <0.05 versus rhabdomyolysis treated with saline at the corresponding times (n = 10). (d) MSC therapy markedly reduced acute tubular necrosis (ATN) scores 72 hours after rhabdomyolysis. (e-h) Treatment with the sham-operated control (Sham + NS and Sham + MSCs) had no effect on renal histopathological parameters (e and f), while MSC therapy markedly improved tubular injury (h), and mice treated with saline showed severe tubular injury (g) (HPFs, ×400, n = 10). HPFs, high-power fields; NS, normal saline.

Mentions: To determine whether MSCs attenuate rhabdomyolysis-induced AKI, we subjected the mice to an intramuscular injection with 50% glycerol solution following water deprivation for 24 hours. We administered MSCs (106 per mouse) six hours after rhabdomyolysis. MSC infusion markedly reduced the levels of serum creatinine (SCr, Figure 1a), blood urea nitrogen (BUN, Figure 1b) and phosphocreatine kinase (CK, Figure 1c) 24, 48 and 72 hours after glycerol injection compared with mice administered saline. Because the maximum AKI was achieved at 72 hours in our study, we selected the 72-hour time point to evaluate kidney injury. Sham mice with an intramuscular injection of NS (Figure 1e) or MSCs (Figure 1f) did not show any significant tubular damage. Rhabdomyolysis (RM) mice receiving normal saline treatment showed tubular necrosis, tubular dilatation and cast formation (Figure 1g). Infused MSCs markedly improved tubular injury (Figure 1h) and lowered the AKI score (Figure 1d) after 72 hours 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)

Mesenchymal stem cells (MSCs) ameliorate rhabdomyolysis (RM)-induced acute kidney injury (AKI). (a-c) Compared with animals treated with saline, infused MSCs significantly reduced serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)) and serum phosphocreatine kinase (CK(c)) levels 24, 48 and 72 hours after RM, and there were no significant changes following treatment of sham mice. *P <0.05 versus sham, #P <0.05 versus rhabdomyolysis treated with saline at the corresponding times (n = 10). (d) MSC therapy markedly reduced acute tubular necrosis (ATN) scores 72 hours after rhabdomyolysis. (e-h) Treatment with the sham-operated control (Sham + NS and Sham + MSCs) had no effect on renal histopathological parameters (e and f), while MSC therapy markedly improved tubular injury (h), and mice treated with saline showed severe tubular injury (g) (HPFs, ×400, n = 10). HPFs, high-power fields; NS, normal saline.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Mesenchymal stem cells (MSCs) ameliorate rhabdomyolysis (RM)-induced acute kidney injury (AKI). (a-c) Compared with animals treated with saline, infused MSCs significantly reduced serum creatinine (SCr (a)), blood urea nitrogen (BUN (b)) and serum phosphocreatine kinase (CK(c)) levels 24, 48 and 72 hours after RM, and there were no significant changes following treatment of sham mice. *P <0.05 versus sham, #P <0.05 versus rhabdomyolysis treated with saline at the corresponding times (n = 10). (d) MSC therapy markedly reduced acute tubular necrosis (ATN) scores 72 hours after rhabdomyolysis. (e-h) Treatment with the sham-operated control (Sham + NS and Sham + MSCs) had no effect on renal histopathological parameters (e and f), while MSC therapy markedly improved tubular injury (h), and mice treated with saline showed severe tubular injury (g) (HPFs, ×400, n = 10). HPFs, high-power fields; NS, normal saline.
Mentions: To determine whether MSCs attenuate rhabdomyolysis-induced AKI, we subjected the mice to an intramuscular injection with 50% glycerol solution following water deprivation for 24 hours. We administered MSCs (106 per mouse) six hours after rhabdomyolysis. MSC infusion markedly reduced the levels of serum creatinine (SCr, Figure 1a), blood urea nitrogen (BUN, Figure 1b) and phosphocreatine kinase (CK, Figure 1c) 24, 48 and 72 hours after glycerol injection compared with mice administered saline. Because the maximum AKI was achieved at 72 hours in our study, we selected the 72-hour time point to evaluate kidney injury. Sham mice with an intramuscular injection of NS (Figure 1e) or MSCs (Figure 1f) did not show any significant tubular damage. Rhabdomyolysis (RM) mice receiving normal saline treatment showed tubular necrosis, tubular dilatation and cast formation (Figure 1g). Infused MSCs markedly improved tubular injury (Figure 1h) and lowered the AKI score (Figure 1d) after 72 hours 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