Abstract
Objective To investigate the protective effects of the exosomes extracted from splenic ischemic preconditioning (sIPC) on renal ischemia-reperfusion (IR) injury.
Materials and methods Splenic ischemic preconditioning(sIPC)was conducted on mice in vivo 24 hours before the start of renal ischemia-reperfusion (IR) injury experiment, and serum exosomes derived from sIPC mice were infused into the mice model of renal ischemia-reperfusion injury. The kidney tissue and serum were collected 24 hours later. The morphological changes and inflammation in ischemia-reperfusion kidneys were determined by hematoxylin-eosin (HE) staining.Then the apoptosis of kidney tissue sections were detected by TUNEL staining, Ki-67 immunohistochemical staining was used to assess the proliferation.In addition, the levels of pro-inflammatory cytokines including TNF-α, IL-1β and SCr in serum were measured by ELISA.
In vitro, we extracted exosomes from mouse spleen fibroblasts pretreated with hypoxia and reoxygenation (H/R) and administered them to mouse renal epithelial cells.Furthermore, for the hypoxia-reoxygenation model of renal epithelial cells, TUNEL and flow cytometry were used to evalutaed cell apoptosis;Then ELISA was used to measure the levels of TNF-α and IL-1β in the cell supernatant, Bax and Bcl-2 were measured by Western Blotting.
Results HE staining showed that the renal injury caused by ischemia-reperfusion attenuated after sIPC. TUNEL staining showed that renal tissue apoptosis was greatly reduced after sIPC or injection of exosomes extracted from splenic fibroblast hypoxia-reoxygenation model. Ki-67 staining showed that the positive rates of IRI+sIPC group, IRI+mSF(H/R)-exo group, IRI+mSF(H/R+PBS)-exo group were close, higher than IRI group but lower than sham group. ELISA test of kidney tissue showed that the serum creatinine, TNF-α and IL-1β induced by IRI decreased with sIPC and addition of the above-mentioned exosomes.In vitro, the exosomes extracted from the hypoxia-reoxygenation model of splenic fibroblasts had the same protective effect on hypoxia-reoxygenated mouse renal epithelial cells model, and this protective effect disappears after the addition of exosome inhibitors.TUNEL and flow cytometry showed that the exosomes reduced the apoptosis. The ELISA test results showed that the levels of TNF-α and IL-1β in the H/R group increased significantly, but decreased due to the splenic fibroblast exosomes treated with starvation.While the exosome inhibitors inhibited the effects of exosomes.Western blot results showed that the Bax expression level of the H/R group increased, and the Bcl-2 decreased.While the starvation-treated splenic fibroblast exosomes decreased the Bax level and increased the Bcl-2 level.
Conclusions The exosomes extracted from splenic ischemic preconditioning exerted a protective capacity to attenuate renal IR injury.
Competing Interest Statement
The authors have declared no competing interest.
