QUERCETIN, A NOVEL EXOSOMES INHIBITOR, ALLEVIATES RENAL TUBULOINTERSTITIAL INFLAMMATION AND FIBROSIS

Abstract BACKGROUND AND AIMS Tubulointerstitial inflammation (TI) and tubulointerstitial fibrosis (TIF) are the common pathological manifestations of all progressive chronic kidney diseases (CKD). Exosomes-mediated cellular communication between tubular epithelial cells (TECs) and macrophages play an important role in the development of TI and TIF. Exosomes from injured kidneys of CKD mice are increased, while inhibition of exosomes secretion prevents renal fibrosis. Hsp90 is known to promote release of exosomes through Skip/Hop. Here, the protective effects against kidney TI and TIF of quercetin, a novel exosomes inhibitor, were studied. METHOD For unilateral ureteral obstruction (UUO), male C57BL/6J mice were subjected to a double-ligating right ureter following a back incision. The UUO mice were randomly classified into four groups: UUO 7d, UUO 7d + Q, UUO 14d, UUO 14d + Q. Quercetin was intra-gastrically administered at a dose of 50 mg/kg daily. For LPS model, mice were administered intraperitoneally with 200μg LPS. The LPS mice were randomly divided into two groups: LPS group, LPS + Q group. The mice within LPS + Q group were intragastrically treated with Quercetin at a dose of 50 mg/kg daily. Mice were euthanized at Day 3 post-LPS injury. In vitro, TECs were treated with LPS or LPS + Quercetin. TECs were co-cultured with Raw264.7 cells in transwell chambers. After 24 h incubation, exosomes from DiI-labeled TECs were released on the other side and were absorbed by macrophages, which were fixed, stained and observed. To prepare TEC-exos, TECs were cultured in 10% FBS in medium for 24 h. And then cells were maintained for 48 h in a medium with 10% exosomes-deprived FBS for exosomes extraction. TEC-exos were used to interfere with macrophages. To investigate the role of Hsp70 and Hsp90 in exosomes generation and secretion, small interfering RNA (siRNAs) and a plasmid containing Hsp70 or Hsp90 expression gene were used to transfect TECs. RESULTS In UUO and LPS mice model, quercetin inhibited the inflammation and fibrosis of the kidney, as illustrated by decreased expression of IL-18, IL-1β and TNF-α in qPCR, reduction of fibrosis area in Masson staining and inhibited expression of collagen Ⅰ and fibronectin in qPCR. The expressions of Hsp70 and Hsp90 were significantly higher in the UUO group and LPS group than in the vehicle group. With the administration of quercetin, the expression of Hsp70 and Hsp90 showed a marked decline. Renal exosomes particles, and expression of exosomes markers (Alix, CD63), were upregulated in UUO and LPS mice models and downregulated in quercetin-treated groups. In vitro, quercetin abolished Hsp70 and Hsp90 upregulation induced by LPS in TECs. Quercetin also reduced LPS-induced increase in exosomes secretion of TECs. Exosomes secretion of TECs was reduced by silencing Hsp70 or Hsp90 by siRNA, while increased by overexpressing Hsp70 or Hsp90 by the plasmid. CONCLUSION We provided evidence that quercetin, an easily available and nontoxic agent, could be applied as a new therapeutic agent for the treatment of TI and TIF via inhibiting the generation and secretion of exosomes.