Apelin/APJ系统在肾脏纤维化过程中对TGF-b/Smads信号通路的调控作用及其机制的研究

Renal fibrosis is the final common pathway that chronic kidney disease (CKD) progresses to end-stage renal diease (ESRD). Renal tubular epithelial mesenchymal transition (EMT) is the important mechanism of renal fibrosis, which is regulated by much cytokine. The TGFβ/Smads signaling pathway is accepted as the main one to induce renal fibrosis. As an endogenous peptide, apelin and its receptor APJ is widely distributed in most tissues. It has been proved that apelin/APJ system promotes hepatic pibrosis, but can play a certain protective effect in myocardial fibrosis. However, there is still lack of reports about the relationship between apelin/APJ system and renal fibrosis. Our previous study showed that the serum level of apelin in uremia patients was much higher than healthy controls. We also observed that apelin was able to inhibit TGFβ1-induced EMT of tubular epithelial cells. In the present study, we will culture human renal tubular epithelial cells, build renal interstitial fibrosis mouse model and apelin deficient mouse model, to investigate the relationship between apelin/APJ system and renal fibrosis from multi-angle. We want to clarify that the apelin/APJ system can suppress renal fibrosis and CKD progression through the important TGFβ/Smads pathway, which would make apelin be deeply researched as a potential medicine for CKD in future.

肾脏纤维化是慢性肾脏病（CKD）进展至终末期肾脏病（ESRD）的共同通路。肾小管上皮细胞-间充质转分化（EMT）是肾脏纤维化发生发展的重要机制，受众多细胞因子的调控。其中TGFβ/Smads通路是公认的主要途径。Apelin与其受体APJ广泛分布于人体组织器官中。现已证实它能促进肝纤维化，抑制心肌纤维化。对于该系统与肾脏纤维化的关系尚缺乏报道。前期研究显示，尿毒症患者血浆Apelin水平显著高于正常人，且Apelin能抑制肾小管EMT。本研究计划采用体外培养人肾小管上皮细胞、单侧输尿管结扎致肾脏纤维化小鼠模型、Apelin基因敲除小鼠模型，通过分子生物学和免疫组织化学等技术，从多层面探讨CKD状态下Apelin/APJ系统的代偿性变化，以及Apelin能否通过抑制经典的TGFβ/Smads途径抑制肾小管EMT，进而减轻肾脏纤维化，延缓CKD进展。

研究背景：肾小管上皮-间充质转分化（EMT）是肾脏纤维化的重要机制，TGFβ/Smads通路是诱导EMT的主要途径。Apelin与其受体APJ广泛分布于肾脏。已证实其促进肝纤维化，抑制心肌纤维化。但与肾脏纤维化的关系尚不清楚。本研究探讨肾脏纤维化时机体Apelin/APJ系统的代偿性变化，以及Apelin能否通过抑制TGFβ/Smads途径抑制EMT，进而减轻肾脏纤维化。.方法:（1）细胞实验：体外培养近端肾小管上皮细胞，给予TGF-β1和/或Apelin孵育。检测上皮和间充质标志物的表达。检测转分化信号分子的活化及表达。加入APJ受体拮抗剂（F13A），重复上述实验。（2）动物实验：构建单侧输尿管结扎小鼠模型（UUO）及假手术模型（Sham），给予生理盐水或Apelin腹腔注射。检测小鼠血浆Apelin变化。取手术侧肾脏，观察纤维化程度，肾脏APJ表达。检测肾小管上皮标志物、间充质标志物和细胞外基质的表达。检测TGF-β/Smads通路的活化和表达。构建Apelin基因敲除小鼠（KO），以野生型小鼠（WT）做对照，分别建立UUO和Sham模型。比较上述标志物表达的差异。.结果：（1）细胞部分：与对照组相比，TGF-β组上皮标志物E-cadherin表达减少，间充质标志物α-SMA表达增多，而TGF-β+Apelin组上述效应受到抑制。与TGF-β组相比，TGF-β+Apelin组活化型Smads（p-Smads2/3）水平降低，抑制型Smads（Smad7）表达增加。加入F13A后Apelin的效应受到显著抑制。（2）动物部分：UUO+盐水组肾脏可见明显间质纤维化，细胞外基质FN、Col-Ⅰ和Col-Ⅲ表达升高；肾小管上皮可见转分化表现，信号分子TGF-β、TβRI受体、p-Smad2的表达升高。而UUO+Apelin组肾间质纤维化程度、细胞外基质和转分化标志物表达量均低于UUO+盐水组。Sham+盐水组和Sham+Apelin组上述标志物的表达处在正常水平。UUO+盐水组肾脏APJ表达升高，但Apelin的表达无明显变化。基因敲除实验显示，KO+UUO组和WT+UUO组小鼠相比，肾脏纤维化程度更严重，EMT表现更显著。.结论：Apelin通过干扰TGFβ/Smads信号通路抑制EMT，进而减轻肾脏纤维化。在肾脏纤维化时，自身APJ表达升高，可代偿慢性肾脏病进展。