Hypoxic Renal Tubular Epithelial Cells-derived Exosome Promoted Peritubular Endothelial Cell Proliferation Via Transfer Of VEGFA In Ischemic Kidney Injury

Background: Acute kidney injury(AKI)is characterized by substantial injury of renal tubular epithelial cells(TECs)and peritubular capillaries(PTCs)rarefaction.Our previous studies revealed that increasing number of exosomes were secreted by hypoxic TECs during AKI.However,the effect of TECs exosomes on the injury and repair of the closely located PTCs remains unknown.The aim of this study was to explore the effect of the TECs exosomes on the PTCs in AKI and the underlie mechanism was investigated.Methods: Ischemia-reperfusion(I/R)injury mice was established and sacrificed at Day 1,3,7.Blood were collected for renal function analysis and kidney tissue sample were harvested for pathologic staining and molecular biology analysis.To explore the effect of TEC exosomes on PTCs,exosomes were purified and characterized from hypoxia TECs,and were administered to HUVEC in vitro.Besides,a transwell co-culture system was conducted to observe the internalization of exosomes.Finally,inflammation and proliferation of PTCs were detected when exosome secretion was inhibited by knockdown of Rab27 a in vivo through lentiviral sh RNA administration in I/R mice.Results: In this study,tubulointerstitial inflammation and microvascular rarefaction were observed in I/R treated kidneys at Day 1 and 3.Tubular injury marker KIM-1,and kidney ICAM-1,VCAM-1,ICAM-1,TNF-α m RNA was induced significantly compared to sham mice.Besides,PCNA is highly expressed in I/R treated kidneys at Day 1 and 3 and increasing proliferation of TECs and PTCs were observed as detected by PCNA staining.Thus,injured TECs and PTCs caused by ischemia lead to extensive cell proliferation in I/R mice.In vitro,exosomes from hypoxia and control HK-2 cells were purified and characterized.Cup-shaped vesicles was found with transmission electron microscopy(TEM)analysis,and nanoparticle-tracking analyses(NTA)revealed exosomes with an average size of 145.9 nm and 156.5 nm respectively from normal and hypoxia HK2.Significant increasing number of exosomes were released by hypoxic TECs compared with controls,as detected by NTA and quantification of exosome markers,Alix,CD63.Exogenous exosomes from hypoxic TECs induced significant proliferation of endothelial cells and upregulation of ICAM-1,VCAM-1,MCP-1,TNF-α expression compared to exosomes from normoxia TECs.Interestingly,we found that VEGFA was significantly up-regulated in the exosomes fractions purified from hypoxia HK-2 as well as kidney from Ischemia-reperfusion(I/R)injury mice.Exosomes from HK-2cells with VEGFA si RNA attenuated cell proliferation induced by exosomes in HUVECs.Transwell co-culture study showed that exosomes travel through membrane and were internalized by HUVEC.Importantly,flow cytometry showed that internalization of exosomes significantly decreased when VEGFR was knock down in endothelial cells.Inhibition of exosome production by knock down of Rab27 a in vivo repressed PTCs inflammation and proliferation after ischemic injury.Conclusion: In conclusion,we have demonstrated that in the setting of I/R-induced AKI,exosomes are released in greater numbers by TECs packed with VEGFA.The exosomal VEGFA can be delivered to ECs of PTCs,provoking their inflammation and angiogenesis.These findings provide a previously unrecognized mechanism of exosome mediated cross-talk between TECs and PTCs in regulating kidney injury and repair after AKI.