Complement-mediated injury of the kidney: New mechanisms and novel therapies

描述（由申请人提供）：最近的研究表明，补体替代途径的激活是大量肾小球和小管间质性肾脏疾病发病机制的核心。然而，目前尚不清楚为什么肾脏如此频繁和独特地易受替代途径介导的损伤。本研究的中心假设是血浆蛋白因子H对于控制肾小球基底膜和受损系膜细胞和小管上皮细胞表面的替代通路激活至关重要。肾内补体对因子H的调节被局部产生的损害因子H功能的蛋白质和全身受损细胞脱落的补体活化微粒所破坏。为了验证这一假设，将追求以下具体目标。目的1)确定哪些调节蛋白控制肾脏补体活化。这一目标的工作假设是，因子H阻止了GBM和受伤的系膜和小管上皮细胞的自发补体活化。我们将使用体外和体内方法来确定哪些肾表面需要因子H来预防补体介导的损伤。我们将使用靶向补体抑制剂将因子H恢复到肾脏内补体活化的位点。目的2)确定膜联蛋白A2如何通过肾内因子H调节补体调节。这一目标的工作假设是，受损肾脏内表达的膜联蛋白A2阻断了因子H的功能，从而允许局部激活替代途径。本实验将利用重组膜联蛋白A2、SiRNA和靶向缺失膜联蛋白A2基因的小鼠，以确定膜联蛋白A2在体外和体内如何促进肾脏细胞表面补体活化。目的3)确定血浆微粒是否损害肾脏内补体调节。这一目标的工作假设是，从受损内皮细胞脱落的微粒导致肾脏内补体活化。这些实验将利用环孢素生成内皮微粒，并测试它们是否对因子H缺乏小鼠造成损伤。将环孢素诱导的微颗粒注射到因子H缺陷小鼠体内，以确定微颗粒是否直接增加替代通路激活并引起肾损伤。最后，我们将测试靶向补体抑制剂cr2 -因子H是否能预防环孢素诱导的因子H缺陷小鼠血管和肾脏毒性。上述方法具有创新性，因为它挑战了目前对因子H如何控制组织补体激活的理解，并解释了为什么在所有器官中，肾脏如此独特地容易受到替代途径介导的损伤。这项资助的研究意义重大，因为它们研究了一系列肾脏疾病发病机制的核心过程，并为这些疾病提供了新的治疗策略。

DESCRIPTION (provided by applicant): Recent work has shown that activation of the alternative pathway of complement is central to the pathogenesis of a large number of glomerular and tubulointerstitial renal diseases. It is not known, however, why the kidney is so frequently and uniquely vulnerable to alternative pathway-mediated injury. The central hypothesis of this grant is that the plasma protein factor H is critical for controlling alternativ pathway activation on the glomerular basement membrane and the surface of injured mesangial cells and tubular epithelial cells. Complement regulation within the kidney by factor H is disrupted by the local production of proteins that impair factor H function and by complement-activating microparticles shed by injured cells throughout the body. To test this hypothesis, the following specific aims will be pursued. Aim 1) Determine which regulatory proteins control complement activation in the kidney. The working hypothesis for this aim is that factor H prevents spontaneous complement activation on the GBM and on injured mesangial and tubular epithelial cells. We will use in vitro and in vivo methods to determine which renal surfaces require factor H to prevent complement-mediated injury. We will use targeted complement inhibitors to restore factor H to sites of complement activation within the kidney. Aim 2) Determine how the protein annexin A2 modulates complement regulation by factor H within the kidney. The working hypothesis for this aim is that annexin A2 expressed within the injured kidney blocks factor H function, thus permitting local activation of the alternative pathway. The experiments in this aim will utilize recombinant annexin A2, SiRNA, and mice with targeted deletion of the gene for annexin A2 in order to determine how annexin A2 promotes complement activation on the surface of cells of the kidney in vitro and in vivo. Aim 3) Determine whether plasma microparticles impair complement regulation within the kidney. The working hypothesis of this aim is that microparticles shed from injured endothelial cells cause complement activation within the kidney. These experiments will utilize cyclosporine to generate endothelial microparticles and test whether they cause injury in factor H deficient mice. Cyclosporine-induced microparticles will be injected into factor H deficient mice to determine whether the microparticles directly increase alternative pathway activation and cause renal injury. Finally, we will test whether CR2-factor H, a targeted complement inhibitor, prevents cyclosporine-induced vascular and renal toxicity in factor H deficient mice. The approach outlined above is innovative because it challenges the current understanding of how factor H controls complement activation on tissues and it provides an explanation for why the kidney, among all of the organs, is so uniquely susceptible to alternative pathway-mediated injury. The studies in this grant are significant because they examine a process central to the pathogenesis of a wide range of renal diseases and provide new treatment strategies for these diseases.