淫羊藿苷在急性肾损伤向慢性肾脏病转化中的雌激素样作用与机制研究

Acute Kidney Injury (AKI) is a prevalent clinical disease, mainly characterized by acute tubular injury. While the pathology of Chronic Kidney Disease（CKD）is known as renal fibrosis. It has been proved recently that severe and repeated tubular injury would inevitably lead to chronic kidney disease. During that process, dysfunction of mitochondrial metabolism plays an important role. Thus, it is pivotal to figure out the way of preventing and treating AKI to CKD transition. It is widely recognized that low estradiol level could ameliorate the development and progression of acute kidney injury, and facilitate renal interstitial fibrosis. But whether Estradiol has a protective effect on the AKI-CKD transition remains unclear, meanwhile, the side effect of estradiol has also restricted its application in the clinical practice. Interestingly, an active ingredient of Epimedium- Icariin has been verified to have the same effect as estradiol does on attenuation of renal fibrosis, but with less side effect. Based on above, this study is aimed to use two classical AKI-CKD transition mouse models（AA1 model and IRI model）and a novel method of co-culture of the renal tubular epithelial cell line (NRK-52E) and renal fibroblast cell line (NRK-49F) to mimic the cross-talk between the tubule and interstitium. Whether the effect of estradiol and Icariin on AKI-CKD is via mitochondrial metabolism is to be discussed.

急性肾损伤（AKI）是临床常见病，其主要病理特征是急性肾小管损伤。慢性肾脏病（CKD）的病理基础是肾纤维化。近来发现反复和严重的肾小管损伤，可导致CKD发生，而线粒体代谢异常在AKI至CKD发展中发挥重要作用。因此预防和扭转AKI向CKD转化成为目前肾脏病研究的重点和热点。雌激素水平低下可加重AKI，雌激素可保护线粒体代谢异常，但雌激素是否具有防治AKI向CKD转化的作用尚未完全阐明，其副作用也限制了该药物在临床的应用。补肾中药仙灵脾的有效成分淫羊藿苷已被证实具有雌激素样作用，并可改善肾纤维化。基于以上，本研究采用马兜铃酸肾病和缺血/再灌注两种经典AKI致CKD模型，动态观察雌激素及淫羊藿苷对AKI向CKD转化的防治作用，并将肾小管上皮细胞与肾间质成纤维细胞共培养，模拟小管与间质的cross-talk，并探究雌激素与淫羊藿苷对肾小管上皮细胞线粒体代谢通路的保护作用。

急性肾损伤（Acute Kidney Injury，AKI）是临床常见病，其主要病理特征是急性肾小管损伤，慢性肾脏病（Chronic Kidney Disease，CKD）的病理基础是肾纤维化。众多研究表明反复和严重的急性肾损伤更会增加慢性肾脏病发病风险，最终不可逆地进展为终末期肾病（End Stage Renal disease，ESRD）。因此预防和扭转AKI向CKD转化成为目前肾脏病研究的重点和热点。雌激素水平低下可加重AKI，但雌激素是否具有防止AKI向CKD转化的作用尚未完全阐明，其副作用也限制了该药物的临床应用。补肾中药仙灵脾的有效成分淫羊藿苷被证实具有雌激素样作用，并可改善肾纤维化，而其对AKI向CKD转化的作用与机制尚未完全阐明。本研究通过构建马兜铃酸肾病和缺血再灌注的经典AKI致CKD模型，予造模前一周给予淫羊藿苷持续灌胃，检测肾功能、肾纤维化等指标；并将肾小管上皮细胞与肾间质成纤维细胞共培养，予马兜铃酸（AA1）刺激肾小管上皮细胞株，加用淫羊藿苷干预，观察肾小管上皮细胞炎症因子分泌和肾间质成纤维细胞株纤维化因子合成情况。并运用CRISPR/Cas9基因编辑技术构建雌激素膜新型受体Gper1敲除小鼠，并获得原代肾小管上皮细胞Gper1敲除细胞，观察淫羊藿苷保护肾小管功能是否通过GPER1起作用。结果发现淫羊藿苷对马兜铃酸肾病模型和缺血再灌注AKI致CKD的两种模型均具有保护作用，可抑制纤维化因子的合成，并减少炎症细胞浸润。通过肾小管上皮细胞和成纤维细胞共培养发现，淫羊藿苷通过保护肾小管上皮细胞抑制炎症因子及促纤维化因子，从而抑制成纤维细胞向肌成纤维细胞转化。而且通过进一步构建Gper1基因敲除鼠，发现淫羊藿苷延缓AKI致CKD转化的作用在基因敲除小鼠和原代肾小管上皮细胞上消失。本研究初步证实淫羊藿苷可通过雌激素膜受体GPER1起到肾小管上皮细胞保护作用，从而缓解急性肾损伤向慢性肾脏病转化。