Targeted treatment of rat AKI induced by rhabdomyolysis using BMSC derived magnetic exosomes and its mechanism

Figures

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  Scheme 1. Exo-SPION treatment using magnetic navigation.

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  Fig. 1. Characterization of SPION and Exo-SPION. (A) Representative TEM images of SPION. Scale, 20 nm. (B) DLS was used to measure the average hydrodynamic size of the SPIONs. (C) Zeta potential of the SPIONs. (D) TEM image of BMSC-SPION showing an enlarged view of endosomes. Scale bar, 500 nm. (E) Morphology of Exo-SPION under TEM scale bars, 100 nm. (F) Representative nanoparticle tracking analysis of Exo-SPION. (G) Exo specific biomarkers (CD81, TSG101, Alix and calnexin) in Exo, Exo-SPION and BMSC whole-cell lysates were analyzed via western blotting. (H and I) Toxicity study of the SPION on BMSCs. CCK-8 assay was used to evaluate the proliferation of BMSCs treated with different concentrations of SPION. (J) The dose dependent uptake of SPION by BMSCs was evaluated by Prussian blue staining. Blue represents SPIONs in BMSC. Scale bar, 500 μm (*P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 μg Fe per mL; n = 6).

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  Fig. 2. Prussian blue staining of the lung, kidney and muscle 3 days after AKI was induced by glycerol. There were 6 rats in the PBS group, Exo group, Exo-SPION group and Exo-SPION+MF group. The red arrow indicates that Fe is highly enriched within the cell. Scale, 50 μm.

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  Fig. 3. Different forms of exosomes protect the muscles. There were 6 rats in the PBS group, Exo group, Exo-SPION group and Exo-SPION+MF group. Control group (n = 3). (A and B) After 3 days of tail vein injection, the levels of LDH and SOD in the serum of AKI rats were measured. (C) Representative micrographs of muscle tissues stained with H&E. Scale bar, 50 μm. (D and E) The serum CK and Mb levels. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the PBS group.

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  Fig. 4. The expression of Bcl2, Caspase3, IL-1β and IL-10 positive cells in the muscles of rats in each group. (A) The expression level in the muscle tissues was showed by IHC. Scale bar, 50 μm. (B) Quantitative analysis of IL-1β, IL-10, Caspase3 and Bcl2 protein expression in muscle of rats in each group. *P < 0.05, **P < 0.01 compared to the control group. #P < 0.05 compared to PBS group.

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  Fig. 5. Different forms of exosomes protect the kidneys. There were 6 rats in the PBS group, Exo group, Exo-SPION group and Exo-SPION+MF group. Control group (n = 3). (A) After 3 days of tail vein injection, the levels of Cre and UREA in the serum of AKI rats were measured. (B) The serum OH-1 and Kim-1 levels. (C) Representative micrographs of renal tissue paraffin sections stained with H&E. Treatment with Exo-SPION+MF strongly alleviated the progression of AKI. Scale bar, 50 μm. (D) Renal tubular injury score. (E) Determination of iron content in the heart, liver, spleen, lung and kidney of rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. #P < 0.05, ##P < 0.01 compared to the PBS group.

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  Fig. 6. Expression of proteins in the kidneys of rats. Compared with those in the control group, the inflammatory and apoptotic factors in the kidneys of the rats significantly increased within 3 days after modeling. (A) Representative images of protein imprinting in the kidneys. (B–E) Protein blotting analysis showed that treatment with exogenous Exo-SPION+MF prevents renal inflammation and apoptosis in AKI rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the PBS group (n = 6).

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  Fig. 7. TUNEL staining of apoptotic cells in renal cortex. Apoptosis positive cells appear green. Normal cells appear blue. The positive cells in the PBS group and Exo group increased significantly, followed by Exo-SPION, and the Exo-SPION+MF group decreased significantly. Scale bar, 50 μm.

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  Fig. 8. The therapeutic mechanism of Exo-SPION on damaged cells. (A) The effect of different forms of Exo on the expression of p-ERK and ERK proteins was determined by western blotting. L6 and NRK-52e cells were treated with NO-Exo, Exo, Exo-SPION or Exo-SPION+MF for 6 hours. (B) Statistical map of p-ERK/ERK expression levels in L6 and NRK-52e cells. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group; #P < 0.05, ##P < 0.01 compared to the NO-Exo group.

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  Fig. 9. Changes in the in vitro WNT/β-catenin signaling pathway. (A) Representative images. (B) Exo-SPION+MF inhibits WNT/β-catenin signaling pathway and reduces renal tubular cell fibrosis. (C, D, G and H) Statistical analysis of WNT and β-catenin protein expression. (E, F, I and J) Statistical analysis of WNT and β-catenin protein expression. GraphPad was used for the statistical analysis of the data. The results are expressed as the mean ± standard deviation, *P < 0.05, **P < 0.01 compared to the control group, #P < 0.05, ###P < 0.001 compared to the NO-Exo group. $P < 0.05 compared to the Exo group.