骨形成蛋白（BMP）受体RGMb在急性肾损伤中的作用及机理研究

Acute kidney injury (AKI) is a common clinical condition that is associated with considerable morbidity and mortality in humans. Renal ischemia reperfusion injury (IRI) is a common cause of AKI. But there is no effective therapy for this devastating clinical condition. RGMb, a member of the repulsive guidance molecule family, has been identified as co-receptor that enhances BMP signaling. Our previous studies showed that RGMb was highly expressed in tubular epithelial cells and involved in the regulation of apoptosis and tubular injury in the kidney after unilateral ureteric obstruction, but these regulations were not observed in renal IRI. Therefore, the role and mechanism of RGMb in renal IRI will be studied in this project. First, in order to investigate the role of RGMb in renal IRI, we have generated renal tubular specific RGMb knockout mice. We will perform renal IRI on these mice and then analyze renal structure and function. Second, in order to study the effect of RGMb on necroptosis which induced by renal IRI, we will examine the expression and activation of RIP1，RIP3 and MLKL, the three key mediators of necroptosis, during IRI in both wild-type mice and RGMb condition knockout mice. Third, we will use renal tubular epithelial cell lines TKPTS and PTCs to study the molecular mechanism by which RGMb regulates necroptosis. Our study will reveal a novel strategy for renal IRI.

肾脏缺血再灌注（IRI）导致的急性肾损伤发病率和死亡率都很高，尚缺乏有效的防治手段。RGMb是骨形成蛋白的辅助受体，在其信号通路调节中发挥重要作用。我们之前的研究显示在UUO损伤中，RGMb损害肾小管上皮的完整性并促进肾小管上皮细胞的凋亡，但是这个现象在肾脏IRI中并没有被观察到。因此本项目研究RGMb在肾脏IRI中的作用及机理。首先，利用肾脏上皮细胞特异性敲除RGMb基因小鼠构建肾脏IRI模型，研究RGMb在肾脏IRI中的作用。其次，分析肾脏IRI中细胞程序性坏死的介导物RIP1、RIP3和MLKL的表达活性变化，研究RGMb与肾脏IRI引起的细胞程序性坏死的关系。最后，利用肾脏上皮TKPTS和PTCs细胞，验证RGMb在细胞程序性坏死中的作用，筛选相关的信号通路，阐明RGMb通过调节细胞程序性坏死影响肾脏IRI的作用机理。本项目的完成将为临床治疗肾脏IRI引起的急性肾损伤提供新思路。

肾小管细胞坏死是急性肾损伤(AKI)的重要组织学特征。程序性坏死是一种受死亡信号调控的死亡方式，它受到混合谱系结构域样激酶（mixed lineage kinase domain-like protein, MLKL）的调控。越来越多的证据表明，程序性坏死在AKI的发生发展中起着重要作用。然而，肾小管细胞是否在体内发生程序性坏死以及坏死通路在AKI期间如何被调控尚不清楚。排斥导向分子(RGM)-b是RGM家族的一员，前期研究表明RGMb在肾小管上皮细胞中高表达，但其在肾脏中的生物学功能仍需进一步研究。在本研究中，我们发现RGMb抑制MLKL的质膜移位及细胞程序性坏死。缺血再灌注损伤(ischemia/reperfusion injury, IRI)时，MLKL在近曲小管基底膜上表达明显升高。与野生型小鼠相比，肾小管上皮细胞特异性敲除RGMb (RGMb cKO)小鼠在近曲小管基底膜上的MLKL表达更高，导致更多的肾小管细胞死亡和更严重的肾功能障碍。在野生型和RGMb cKO小鼠中，用坏死抑制剂Necrostatin-1或GSK’963治疗可以降低近曲小管基底膜上MLKL的表达，改善IRI引起的肾脏损害。综上所述，结果表明近端小管细胞程序性坏死在AKI中起重要作用，RGMb通过抑制MLKL质膜移位和近端小管细胞程序性坏死来保护AKI。