Effect and mechanism of targeted delivery of VEGF and CD47 dual-modifiedexosomes in treatment of acute kidney injury induced by heat stroke in rats in the desert dry hot environment.

Objective To develop an effective method for delivering VEGF and CD47 double-modified exosomes to treat renal damage induced by heat stroke so as to reduce and repair renal damage. Methods A plasmid fusion-expressing VEGF and CD47 targeting renal injury was constructed, transfected into rat bone marrow derived mesenchymal stem cells (BMMSCs), and then fusion-exosomes were isolated and extracted. Transmission electron microscopy, nanoparticle tracking analysis, and Western blotting were used to identify the obtained exosomes. Rats were intravenously injected with 200 μg of DiD-labeled unmodified exosomes, VEGF-modified exosomes and VEGF-CD47 double-modified exosomes, respectively, through the tail vein, and the effects of exosomes on the kidneys were detected and analyzed using a small animal in vivo imaging instrument. A total of 60 SD rats were randomly divided into 6 groups, with 10 rats in each group, that is, blank control group (group A), heat stroke-induced renal injury model receiving PBS in 12, 24 and 36 h after modelling (group B), empty plasmid group (group C), Exos group (group D), ExosVEGF group (group E) and ExosVEGF-CD47. Kidney tissue and blood samples were collected in 72 h after 3 times of treatment. Pathological changes in kidney tissue were observed at the tissue level and the damage were scored. Changes in serum blood urea nitrogen (BUN) and serum creatinine (Scr) levels were detected to evaluate the therapeutic effect. Western blotting and qRT-PCR were used to analyze the expression of the pro-inflammatory factors TNF-α and NF-κB, the proliferation regulatory signaling molecules Ki67, FGF2, pAMPK and pERK, and the fibrosis regulatory molecule FGF23, in order to comprehensively analyze the effects on proliferation and inhibition of fibrosis. Results BMMSCs and ExosVEGF-CD47 were successfully isolated and characterized, and a rat model of acute kidney injury was effectively constructed. Higher fluorescence intensity was found in the kidney tissue of the ExosVEGF-CD47 group than the Exos-Ctrl group and ExosVEGF group (P<0.05). In 72 h after treatment, the ExosVEGF-CD47 group had significantly lower serum BUN and Scr levels (P<0.0001), and notably lower Tubular casts score (P<0.0001), decreased levels of pro-inflammatory factors TNF-α and NF-κB (P<0.0001), up-regulated Ki67 and FGF2 expression (P<0.05), and down-regulated FGF23 expression (P<0.0001) when compared with the AKI+Exos group and AKI+ExosVEGF group. Conclusion VEGF and CD47 show promise in targeting acute kidney injury induced by heat stroke, effectively mitigate damage and facilitate repair, which may be due to exosome-mediated inhibition of renal tissue inflammation, promotion of proliferation, and inhibition of fibrosis.