Low‐energy shock wave pretreatment recruit circulating endothelial progenitor cells to attenuate renal ischaemia reperfusion injury

Figures

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  Figure 1. Pretreatment with LESW attenuated renal injury and promoted kidney repair. A‐C, BUN, SCr, and Cys C were evaluated at 4, 12, 24 h, and 3, 7 d after reperfusion in the four groups. D, Representative images of H&E staining preformed on sections of kidneys at 24 h after reperfusion in the four groups. E, Histopathological scoring of kidney at 24 h after reperfusion in the four groups. F, Representative images of TUNEL, PCNA, and CD34 staining in kidneys at 3 d after reperfusion in the four groups. G‐I, Semi‐quantitative analysis of TUNEL‐positive cells, PCNA‐positive cells, and MVD CD34 were performed by using ImageJ software. Scale bar = 50 μm. *P < .05 (LESW, IRI, LESW + IRI vs Sham); # P < .05 (LESW + IRI vs IRI); Δ P < .05 (IRI vs LESW). BUN, blood urea nitrogen; Cys C, cystatin C; H&E, haematoxylin and eosin; IR, ischaemia reperfusion; LESW, low‐energy shock wave; MVD, microvessel density; PCNA, proliferating cell nuclear antigen; SCr, serum creatinine; TUNEL, terminal transferase‐mediated deoxyuridine triphosphate nick‐end‐labelling

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  Figure 2. LESW increased the number of endothelial progenitor cells (EPCs) in kidney tissues. A and B, The typical EPCs phenotype of isolated EPCs in the kidney was confirmed by CD34, CD133, and VEGFR2 by flow cytometry in the four groups. Representative flow cytometry histograms of isolated EPCs. C, Quantitative analysis of CD34+/CD133+/VEGFR2+ cells in the four groups. *P < .05 (LESW, IRI, LESW + IRI vs Sham); # P < .05 (LESW + IRI vs IRI); LESW, low‐energy shock wave; VEGFR2, vascular endothelial growth factor receptor 2

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  Figure 3. Characterization of isolated circulating endothelial progenitor cells (EPCs). A, Cells adhered to culture plates at day 4. Spindle‐like cells appeared at day 9. Typical cobblestone‐like cells appeared at day 15. Cells emerged after day 17 of cultivation. B, Representative images of immunofluorescent staining using EPCs specific markers CD31/CD133/CD34/VEGFR2 and nuclear marker DAPI. C, Dil‐Ac‐LDL and FITC‐UEA‐1 uptake assay showed the both Dil‐Ac‐LDL/FITC‐UEA‐1‐positive cells, which indicated that the cultured cells were EPCs. D, Representative flow cytometry histograms of cultured EPCs. Scale bar = 50 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; Dil‐Ac‐LDL, 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine acetylated low‐density lipoprotein; FITC‐UEA‐1, fluorescein isothiocyanate Ulex europaeus agglutinin‐1; VEGFR2, vascular endothelial growth factor receptor 2

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  Figure 4. The distribution of Dil‐C18(5)‐DS labelled circulating endothelial progenitor cells (EPCs). A, Representative flow cytometry histograms of Dil‐C18(5)‐DS label ratio. B, Representative fluorescence images of kidneys detected by in vivo imaging system. C, Quantitative analysis of the retention of the Dil‐C18(5)‐DS labelled circulating EPCs in kidneys at 24 h after reperfusion in the four groups. *P < .05 (LESW + IRI vs Sham); # P < .05 (LESW + IRI vs IRI). LESW, low‐energy shock wave

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  Figure 5. Cell tracking in rats injected with CM‐Dil labelled circulating endothelial progenitor cells (EPCs). A, Representative images of CM‐Dil labelled isolated circulating EPCs. B, Representative images of CD133+/CM‐Dil+ cells in kidneys at 24 h after reperfusion in the four groups. Scale bar = 50 μm. LESW, low‐energy shock wave

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  Figure 6. The expression of SDF‐1 and CXCR7 in four groups. A and B, SDF‐1 levels of kidneys and serum at different times after reperfusion in the four groups. C‐E, Relative abundance of SDF‐1/GAPDH and CXCR7/GAPDH were quantified according to Western blots at 24 h after reperfusion in the four groups. F, Representative images of SDF‐1 staining in kidneys at 24 h after reperfusion in the four groups. G, Semi‐quantitative analysis of SDF‐1 immunostaining proportional area. Scale bar = 50 μm. *P < .05 (LESW, IRI, LESW + IRI vs Sham); # P < .05 (LESW + IRI vs IRI). LESW, low‐energy shock wave

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  Figure 7. The renal protective effect of pretreatment with LESW was blocked by CCX771. A‐C, BUN, SCr, and Cys C were evaluated at 24 h after reperfusion in the three groups. D, Representative images of H&E, TUNEL, PCNA, and CD34 staining in the kidneys at 3 d after reperfusion in the three groups. E‐H, Histopathological scoring of kidney, Semi‐quantitative analysis of TUNEL‐positive cells, PCNA‐positive cells, and MVD. Scale bar = 50 μm. *P < .05 (IRI, LESW + IRI + CCX771 vs LESW + IRI). BUN, blood urea nitrogen; Cys C, cystatin C; EPCs, endothelial progenitor cells; H&E, haematoxylin and eosin; LESW, low‐energy shock wave; MVD, microvessel density; PCNA, proliferating cell nuclear antigen; SCr, serum creatinine; SDF‐1, stromal cell–derived factor‐1; TUNEL, terminal transferase‐mediated deoxyuridine triphosphate nick‐end‐labelling

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  Figure 8. Inhibition of CXCR7 prevented the recruitment and migration of circulating endothelial progenitor cells (EPCs). A and B, Representative flow cytometry histograms of isolated EPCs in kidneys and quantitative analysis of CD34+/CD133+/VEGFR2+ cells in the three groups. *P < .05 (IRI, LESW + IRI + CCX771 vs LESW + IRI). C, Representative fluorescence images of kidneys detected by in vivo imaging system. D, Quantitative analysis of the retention of the Dil‐C18(5)‐DS labelled circulating EPCs in kidney at 24 h after reperfusion in the three groups. E, The migrated ability of EPCs following stimulating with SDF‐1 (0‐200 ng/mL) without or with CCX771. Representative images were shown. F, Quantitative analysis of circulating EPCs migration. Scale bar = 50 μm. *P < .05 (10, 25, 50, 100, 200, 200 + CCX771 vs control and 0); + P < .05 (50, 100, 200 vs 10, 25, and 200 + CCX771); # P < .05 (100, 200 vs 50); & P < .05 (200 vs 100). LESW, low‐energy shock wave; SDF‐1, stromal cell–derived factor‐1