外泌体介导上皮间质交流在积水肾急性灌注压力负荷后肾纤维化中的作用及机制研究

The intraoperative perfusion pressure load is considered to be the main trigger factor of aggravated renal interstitial fibrosis during upper urinary lithotripsy, which may lead to further impaired renal function in hydronephrosis, but the mechanism is poorly understood. Exosome mediates cells communication play an important role in fibrosis. Our previous studies found that the expression of TGF-β1 was up-regulated in hydronephrosis induced by irrigation pressures in vivo，and TGF-β1 stimulated exosome secretion of renal tubular epithelial cells, which can activate fibroblast to myofibroblast transition; in addition, inhibition of microRNA-21 expression in exosome retarded fibroblasts to myofibroblasts transition, up-regulated the expression of Spry1, and inhibited ERK signaling pathway. Therefore, we hypothesize that epithelia-mesenchymal communication mediated by exosome is key point to regulate kidney fibrosis induced by acute irrigation pressures in hydronephrosis. This project will establish pressure load damage models in animals and cells, to elucidate the role and mechanism of epithelia-mesenchymal communication mediated by exosomes-delivered microRNA-21 on kidney fibrosis induced by irrigation pressures in hydronephrosis, thus providing targets for prevention and treatment.

上尿路腔内碎石术后患肾间质纤维化加重，可致积水肾功能进一步受损，术中液体灌注压力负荷被认为是其主要促发因素，但其机制待阐明。外泌体（exosome）介导细胞间交流在组织纤维化中扮演重要作用。本课题组前期研究发现急性灌注压力负荷积水肾后TGF-β1表达显著升高；而且TGF-β1刺激肾小管上皮细胞分泌的外泌体激活间质成纤维细胞向肌成纤维细胞转变，抑制外泌体中microRNA-21表达可抑制此转变，同时上调Spry1基因和抑制ERK信号通路。据此，我们认为外泌体介导上皮与间质细胞交流是积水肾急性灌注压力负荷后肾纤维化进展的关键所在。本研究拟建立动物积水肾灌注压力负荷损伤模型和细胞压力负荷的肾纤维化损伤模型，阐明外泌体运载microRNA-21介导上皮与间质交流在积水肾灌注压力负荷后肾纤维化进展中的作用及机制，为积水肾灌注压力负荷后肾间质纤维化提供防治靶点。

肾及输尿管结石是泌尿系统最为常见的疾病之一，结石导致的梗阻往往会造成肾输尿管积水及肾组织纤维化。腔内手术在解除积水肾梗阻的同时，肾盂内灌注压力的急剧升高除引起患侧肾脏的急性损伤外，也是导致术后患肾间质纤维化加重的关键因素。然而其具体机制尚未阐明。我们首先构建了动物及细胞积水肾液体灌注压力负荷损伤模型，并对组织损伤及纤维化进行了检测，结果证实：积水及压力负荷均可导致肾组织损伤及纤维化，且积水越严重的肾脏越容易受到液体灌注压力负荷的影响。为探究外泌体在该模型中发挥的重要作用，我们对肾小管上皮细胞分泌的外泌体进行检测，结果表明：TGF-β1刺激及压力灌注压力负荷可进一步刺激肾小管上皮细胞分泌外泌体，且肾小管上皮细胞来源的外泌体可诱导成纤维细胞增殖及向肌成纤维细胞转化。为明确外泌体发挥作用的具体机制，我们后续进行了外泌体提取、鉴定、miRNA测序及内容物的过表达及抑制相关实验。同时，结合了体内外实验，将提取到的肾小管上皮细胞来源的外泌体通过尾静脉注射进入动物模型体内，进一步明确外泌体了发挥作用的具体机制，即：肾小管上皮细胞来源的外泌体内富含miR-21及miR-23a等内容物，其可通过靶向成纤维细胞的PTEN及SnoN基因，调控Akt及Smad通路，进而促进成纤维细胞的增殖活化，最终导致肾纤维化的进展。此外，我们构建了Rab27a基因敲除鼠，并进行相关实验，最终也得到了相同的实验结论。本项目所获的研究成果不仅可以在分子水平深化对急性肾盂压力升高导致的急、慢性肾损伤的认识，还可进一步丰富肾纤维化的发生理论，为临床积水肾肾功能保护提供理论依据，为围手术期灌注压力负荷所致积水肾纤维化分子干预提供新思路。