从水通道蛋白1介导的EMT/MET平衡探讨“肾主水”的分子机制

TCM zangxiang Theory deams that "kidney governs water", that is, kidney qi is in charge of regulating the whole body water metabolism. Kidney yang deficiency is the common syndrome of patients with chronic kidney disease. The progression of kidney injury is in the performance of renal interstitial fibrosis, closely associating with the imbalance of EMT/MET. The AQP1-mediated cell migration plays an important role in kidney injury repair. Our previous studies have shown that kidney yang deficient rat was with the dysfunction of HPA axis, the decreased expression of AQP1 in kidney tissue, the significantly characteristic of renal interstitial fibrosis, and the imbalance of EMT/MET. Kidney warming prescriptions could increase the expression of AQP1, promote cell migration and mitigate the renal fibrosis. Thus, we speculate that HPA axis could regulate the expression of AQP1 to adjust the migration of renal tubular epithelial cells, to maintain the balance of EMT/MET and to inhibit renal interstitial fibrosis. It may be the important biological basis of "kidney governs water". This study intends to use the experiments in vitro combined with that in vivo, to observe the changes of HPA axis, the expression of AQP1 and the process of EMT/MET in AQP1 gene deletion mouse. Under the guidance of TCM zangxiang Theory using the kidney warming prescriptions to observe the interference effect, and to preliminary elucidate the function of the AQP1-mediated EMT/MET balance in renal interstitial fibrosis. This study will be beneficial to comprehensive understand the essence of " kidney governing water " and provide the basis for kidney warming prescriptions treating chronic kidney disease.

中医藏象理论认为“肾主水”，即肾气主司全身水液代谢。慢性肾脏病患者常见肾阳虚证，肾损伤进展表现为间质纤维化，与EMT/MET失衡密切相关，而AQP1介导的细胞迁移在肾损伤修复中起着重要作用。我们前期研究显示，肾阳虚证大鼠HPA轴功能紊乱，肾组织AQP1表达降低，间质纤维化明显，EMT/MET失衡；温肾药可上调AQP1表达，促进细胞迁移，减轻肾纤维化。由此我们推测，HPA轴通过调控肾脏AQP1表达，调节肾小管上皮细胞的迁移，维护EMT/MET平衡，阻抑肾间质纤维化，这可能是“肾主水”的重要生物学基础。本研究拟体内外实验相结合，观察AQP1基因缺失小鼠HPA轴以及肾组织AQP1、EMT/MET等变化，并采用温肾药“以方测证”，观察其干预效应，初步阐明AQP1介导的EMT/MET平衡在肾阳虚证肾间质纤维化中的作用。该研究有利于全面认识“肾主水”的本质，并为温肾药治疗肾阳虚证慢性肾脏病提供依据。

藏象理论认为“肾主水”，即肾气主司和调节全身水液代谢。前期研究表明，HPA轴可能通过激素调控 AQP1 表达，调节肾小管上皮细胞迁移减轻肾纤维化，维护EMT/MET平衡。这可能是“肾主水”的重要生物学基础。因此本项目组拟通过体内外实验，观察AQP1-/-小鼠HPA轴及AQP1、EMT/MET等变化，并采用肾气丸“以方测证”，初步阐明AQP1介导的EMT/MET平衡在肾阳虚证肾间质纤维化中的作用。.主要研究内容：①采用腺嘌呤诱导和AQP1敲除制备小鼠肾阳虚证肾间质纤维化模型，评估AQP1在肾阳虚证肾间质纤维化模型中的作用，观察两种模型小鼠肾功能、肾纤维化及病理损伤等情况；采用AQP1 RNAi慢病毒感染HK-2细胞，观察AQP1沉默后HK-2细胞EMT相关转录因子、mRNA及蛋白表达情况。②采用肾气丸“以方测证”，观察小鼠一般症状、HPA轴功能、肾功能等改善情况，明确肾气丸的治疗效果；同时采用含药血清干预TGF-β1诱导的HK-2细胞上皮间质转分化模型和AQP1沉默后的HK-2细胞模型，确定肾气丸干预后EMT/MET的转化及AQP1表达变化，明确肾气丸对“HPA轴-AQP1-EMT/MET平衡-肾间质纤维化”的干预机制。.结果显示：①三月龄AQP1敲除小鼠HPA轴功能降低，出现自发性肾阳虚症状与肾纤维化病变，与腺嘌呤75mg/kg诱导14d的小鼠表现出相似的肾阳虚证和病理损伤。②小鼠AQP1敲除及HK-2细胞AQP1沉默后，均表现出EMT/MET失衡。AQP1可能是EMT发生发展中的关键靶点之一。③肾气丸可显著上调AQP1表达，改善HPA轴功能、肾阳虚一般体征及EMT/MET失衡，延缓肾间质纤维化进程。.以上结果表明，肾气丸通过HPA轴调控AQP1表达维护EMT平衡，从而改善肾间质纤维化，初步揭示了“HPA轴-AQP1-EMT/MET平衡-肾间质纤维化”是“肾主水”的分子机制之一。