备解素在肾缺血再灌注损伤及纤维化中的保护作用及机制

Renal ischemia reperfusion injury (IRI) is a main cause of acute kidney injury (AKI), and may lead to interstitial fibrosis/tubular atrophy (IF/TA). The 50% mortality rate of patients with AKI has not changed in the last 5 decades. We have demonstrated IRI mediated up-regulation of caspase-3 expression, inflammation and apoptosis effects, which were down-regulated by leukocyte depletion and treatment with small interfering RNA (siRNA) targeting caspase-3. Latterly, we showed that a novel erythropoietin-derived peptide (CHBP) also improved renal IRI by inhibiting the same outcome measures. .Properdin is the only positive regulator of the alternative pathway (AP) in complement activation. It stabilizes C3bBb, and directly binds to proximal tubular epithelial cells (PTECs), thereby mediating proteinuric renal damage. Properdin knockout also ameliorated renal IRI that was accelerated by knockout of negative regulators of the AP, decay-accelerating factor(DAF)/CD59 and membrane-bound protein Crry. Properdin also effectively recognizes and binds to apoptotic cells through complement-independent mechanisms. Properdin tagging of apoptotic cells facilitates rapid elimination via phagocytosis to avoid inflammatory and autoimmune reactions. Our pilot study revealed that renal IRI was worsened by properdin knockout, which has not been reported before. In addition, there have been no reports describing the interaction of properdin with caspase-3 or properdin with autophagy. We hypothesize that apoptotic cell clearance is compromised by properdin knockout, and affect inflammation and immunity. .This proposed project will be performed in four phases using in vivo and in vitro models with/without properdin knockout: .1. To establish dynamic changes of properdin, in association with caspase-3, in the context of autophagy, apoptosis and inflammation, at both early and later stage of renal IR injury; .2. To dissect the underling mechanisms of properdin using different cells (PTECs, neutrophils, T cells and fibroblasts) challenged by hypoxia/reoxygenation; .3. To modulate properdin in renal IR injury by caspase-3 siRNA and CHBP. .4. To disclose the gene profiling and signalling pathways involving in renal IR injury and fibrosis related to properdin, to validate new mechanistic insights that will benefit diagnosis and intervention.

急性肾损伤诊治困难，死亡率居高不下。缺血再灌注损伤(IRI)是急性肾损伤的主要病因，并可致纤维化。我们的研究表明IRI激活CAPS3酶，增加炎症、凋亡和纤维化；CAPS3小干扰核苷酸和促红素衍生肽抑制该酶，减轻肾IRI（面上基金81170689）。备解素（P，旁路途径阳性调节因子）激活补体，介导蛋白尿性肾损伤；并标识凋亡、坏死细胞，促进吞噬清除。有预实验显示P敲除小鼠肾IRI加重，是否与CAPS3或自噬等相关，尚无文献报道。我们推测P敲除有损凋亡细胞被吞噬清除，增强炎症及免疫反应。本项目拟用有无P敲除的体内、外模型，1.观察早晚期肾IRI中P的动态变化，与CASP3、自噬、凋亡、炎症及免疫反应的关系；2.培养中性粒细胞、肾小管细胞和T细胞等剖析P的作用机制；3.再用CASP3小干扰核苷酸和促红素衍生肽干预P；4.分析与P相关的基因谱及信号通路，验证新机制，改善急性肾IRI和纤维化的的防治。

急性肾损伤（AKI）是影响人们健康的全球性疾病，缺乏有效的特异性治疗。备解素是补体替代途径激活中唯一的阳性调节因子，在肾缺血再灌注（IR）所致的AKI中发挥重要作用。新近研究表明备解素还具有模式识别分子（PRM）的功能，通过标记凋亡T淋巴细胞介导其被巨噬细胞吞噬清除。本研究深入解析了备解素在体内外肾IR相关模型中的表达、功能及与EPOR/βcR的互动。 .我们建立了野生型（WT）小鼠肾IR时点模型；促红细胞生成素（EPO）衍生线肽/环状（HBSP/CHBP）治疗的WT和备解素敲除（PKO）小鼠72 h肾IR模型；和肾IR 48 h、2 w及8 w模型；以及肾小管上皮细胞、巨噬细胞的IR相关损伤模型。.在肾IR早期多时点模型中，随再灌注时间增加备解素表达逐渐升高并在48 h时达到高峰，与肾功能及结构损伤、HMGB1和活性caspase-3蛋白表达呈显著正相关。备解素蛋白和mRNA表达分别在48 h 和72 h达峰值，提示其mRNA和蛋白的调节存在差异。我们的实验结果表明PKO小鼠肾IR 72 h后肾组织损伤显著高于WT小鼠，提示在IR修复期备解素可能作为PRM标记损伤细胞以利吞噬清除。另外，两种小鼠在IR后72 h时肾EPO受体（EPOR）及组织保护受体EPOR/βcR表达均显著升高，并在PKO肾表达更高。提示肾IR损伤越重EPOR/βcR的表达越高以有效地启动修复。HBSP为EPOR/βcR的配体，逆转了WT和PKO小鼠肾IR 72 h模型的肾损伤，治疗后EPOR水平仅在WT小鼠下降，而EPOR/βcR仅在PKO小鼠表达降低。同时，CHBP/HBSP治疗下调了48 h和2 w模型中IR肾脏中高表达的备解素水平。.体外肾小管上皮细胞TCMK-1和巨噬细胞RAW 264.7在H2O2刺激后备解素表达亦升高，但同时使用备解素小干扰（siRNA）干预以上细胞后，HMGB1和活性caspase-3表达进一步升高。更重要的是体外培养原代WT及PKO肾小管细胞（TECs）显示TECs表达的备解素，而不是血清来源的备解素，标记凋亡TECs介导其被吞噬。.本研究揭示了肾小管细胞局部产生的备解素在肾脏IR及相关损伤和修复过程中通过标记损伤细胞包括凋亡小管细胞介导其被吞噬，以利炎症清除中所发挥的PRM作用，对于阐明IR所致AKI及修复的机制及锁定诊疗靶点有重要意义。