Effect of hypoxia on exosomes in renal tubular epithelial cells

Abstract

Objective To explore the effect of hypoxia on exosomes secreted by renal tubular epithelial cells and the function of exosomes in chronic kidney diseases. Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normoxia(21% O2) or hypoxia(1% O2) for 48 h was collected and centrifuged gradiently to harvest exosomes. Exosomes were identified and compared by transmission electron microscope, nanoparticle tracking analysis, Western blotting and measurement of the protein concentration.(2) Primary peritoneal macrophages of rats were co-cultured with exosomes in different concentrations(1, 10, 50, 100, 300 mg/L). The expression of interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), inducible nitric oxide synthase(iNOS) in cells and supernatant were separately detected by quantitative real-time PCR(qRT-PCR) and ELISA, and the expression of phospho(p)-STAT/STAT and suppressors of cytokine signaling 1(SOCS1) in macrophages was detected by Western blotting. At last, the expression of inflammatory microRNAs(miR) in exosomes was measured by qRT-PCR. Results (1) The vesicles harvested by gradient centrifugation were less than 150 nm and expressed CD63 which was characteristic of exosomes. Hypoxia had no effect on the morphology of exosomes, but stimulated their secretion.(2) Hypoxic exosomes dose-dependently improved the expression of IL-6, TNF-α, iNOS in macrophages polarized by lipopolysaccharide(LPS) and increased the expression of p-STAT while decreased the expression of SOCS1(P <0.01). MicroRNAs referred to inflammation such as miR-155 and miR-27a increased in hypoxic exosomes compared to that in normoxic exosomes(P <0.05). Conclusions Hypoxia makes exosomes promoted the polarization of macrophages to M1, which may account for the microinflammation in chronic kidney diseases. Key words: Anoxia; Inflammation; Epithelial cells; Kidney tubules; Exosomes; Macrophages; microRNAs