同型半胱氨酸通过RNA-m6A修饰调控血小板ADP受体表达加重急性肾损伤

Acute kidney injury (AKI) is a major renal disease associated with significant mortality and the development of chronic kidney disease and end-stage renal disease. However, current treatment options are extremely limited. Homocysteine (Hcy) is the important intermediate product of methionine cycle, which provides methyl groups in vivo. High Hcy level results in the reduction of methyl donors and hypomethylated cells. Previous experiments have proved Hcy exacerbates ischemia-reperfusion induced AKI and microthrombus in glomerulus by pathological staining. Otherwise, we also found the expression of platelet receptor P2Y1 was up-regulated, the stability of gene was increased and the total m6A modification ratio was decreased. Therefore, we proposed the hypothesis that Hcy promoted the expression of platelet ADP receptor through RNA-m6A modification to aggravate AKI injury. In order to confirm the above hypothesis, this project intends to carry out 1) Hcy can aggravate AKI injury and promote platelet activation; 2) Hcy regulates the methylation of ADP receptor mRNA and explores the proteins that regulate the methylation of mRNA; 3) Hcy aggravates AKI injury through ADP receptor. This study will further reveal the role and mechanism of Hcy in AKI and provide new targets for clinical prevention and treatment of AKI.

急性肾损伤(AKI)增加住院患者死亡率与致残率，也是慢性肾衰竭的重要原因，但临床治疗手段极为有限。同型半胱氨酸是体内提供甲基的甲硫氨酸循环的中间产物，高Hcy水平导致甲基供体减少，细胞呈现低甲基化。我们前期实验发现Hcy可加重缺血再灌注诱导的AKI损伤，病理染色显示肾脏存在微血栓，血小板受体P2Y1表达上调，mRNA稳定性升高，m6A修饰比例下降。由此我们提出假设：Hcy通过RNA-m6A修饰促进血小板ADP受体表达加重AKI损伤。为证实上述假设，本项目拟行1）Hcy可加重AKI损伤并促进血小板活化；2）Hcy调控ADP受体mRNA甲基化并探索调控mRNA甲基化修饰的蛋白；3）Hcy通过ADP受体加重AKI损伤。本研究将进一步揭示Hcy在AKI中的作用及机制，为临床预防和治疗AKI提供新的靶点。

急性肾损伤（AKI）存在血流动力学异常，局部炎症反应启动凝血过程，血管内微血栓形成，改善全身循环后肾脏局部仍“无复流”，组织持续缺氧，肾脏发生不可逆损伤。研究发现血小板在微血栓形成和炎症中发挥重要作用。临床数据显示，心血管疾病，脑卒中与深静脉血栓均存在血小板功能异常，而高同型半胱氨酸血症（HHcy）是上述疾病的关键危险因子。我们先前的研究发现HHcy加重顺铂引起的急性肾损伤，HHcy可能通过影响血小板的功能参与此过程。HHcy是甲硫氨酸循环一碳单位利用的关键抑制因子，因此本项目的主要研究内容是探索HHcy调控血小板功能加重肾损伤的机制。研究期间我们发现（1）HHcy加重了缺血再灌注急性肾损伤，抑制血小板活化逆转了HHcy加重的肾损伤；（2）HHcy改变了血小板mRNA甲基化修饰状态，m6A修饰水平降低。HHcy引起的m6A的修饰状态变化主要涉及细胞骨架，细胞粘附，免疫细胞激活等功能和通路上调，内皮细胞分化发育，内皮细胞生长因子以及脂肪酸代谢和分解相关功能和通路的下调；（3）HHcy以不依赖血小板生长因子（TPO）的方式通过GH-GHR-PI3K通路促进巨核细胞的发育，引起循环血小板数量升高；（4）HHcy通过降低Nr5a1启动子区域组蛋白H3K4的甲基化水平，下调Nr5a1表达，同时降低睾酮合成酶的水平，导致睾酮水平下降。项目的研究结果表明血小板数量和功能异常是HHy引起肾脏过度损伤的重要机制。HHcy通过血小板RNA-m6A修饰影响血小板功能，在HHcy水平下，血小板表现为与免疫细胞的粘附能力增强等特征。同时HHcy促进了巨核细胞向血小板的发育，增加了血小板的数量。另外，HHcy引起的睾酮下降导致血小板活性增加也是HHcy相关肾损伤和心脑血管疾病的重要发病机制。因此，针对血小板的治疗是有效防治HHcy相关疾病的重要靶点。