Novel Complement-targeted treatment strategies in Renal Disease

项目摘要/摘要 肾小球微血管是调节失调或病理性补体的常见靶点(C) 激活。这与包括狼疮在内的一系列肾小球疾病的发病机制有关。 肾炎、膜增生性肾小球疾病、感染后肾小球肾炎，以及最近的 非典型溶血性尿毒症综合征(AHUS)和C3肾小球疾病。因此，制定战略以 在C依赖性肾小球疾病中，最大限度地减少C级联的激活可能是有希望的。 我们研究的长期目标是通过UP减轻C介导的肾小球损伤的严重程度 调节衰变加速因子(DAF)的表达，它是C激活的关键控制因素，通过 防止自然不稳定的C3和C5转换酶的组装和加速衰退，这些转换酶放大 经典的和替代的补体激活级联。因为目前还没有药理DAF 诱导剂，我们试图确定DAF表达的上调调控因子，其活性可以通过使用现有的 药理策略或药剂我们鉴定了血红素加氧酶(HO)-1，它是血红素的诱导酶 降解为一氧化碳(CO)和胆绿素，成为肾小球中的DAF调节剂。我们证明了这一点 针对内脏肾小球上皮细胞(也称为足细胞)的HO-1过度表达上调 DAF和减少碳沉积和损伤程度。由于它们的非复制性终末分化 在自然界中，足细胞特别容易受到C介导的损伤，而它们的丢失被证明是一个关键 肾小球疾病进展的决定因素。 HO-1上调足细胞DAF的机制尚不清楚。同样未知的是程度 其中，HO-1上调DAF在减轻C介导的足细胞损伤中起关键作用。建议进行的研究 通过追求以下具体目标来解决这些问题： 1)鉴定大鼠足细胞中顺式阳性反应DAF启动子元件 表达HO-1，并检测HO反应产物CO作为特异性DAF激活剂的作用 启动子转录因子。这一目标将检验足细胞一氧化碳产量增加的假设 HO-1过表达通过CO反应顺式作用正调控元件上调DAF表达 在与Sp1转录因子结合位点相对应的DAF启动子上。 2)证明DAF表达增加是HO-1缓解HO-1的关键机制 补体依赖型足细胞损伤。这一目标将检验这样的假设，即在足细胞中过度表达 HO-1、DAF介导C依赖损伤的减轻 AIM I中的实验方法包括使用来自野生型的培养足细胞 (WT)大鼠和缺乏HO-1的大鼠，我们用锌指核酸酶(ZFN)驱动的HO-1基因敲除- 出去。在这些细胞中，HO-1的过度表达和HO反应产物CO对转录的影响 将评估通过Sp1转录因子对DAF启动子的调控。 AIM II中的实验方法包括使用抗体介导的C依赖的大鼠模型 足细胞损伤类似于人类膜性肾病，将在足细胞- 我们利用睡美人(SB)转座子介导的转基因产生的靶向性HO-1过表达 它们要么是DAF完整的，要么是DAF不足的。 来自拟议研究的观察结果预计将产生积极的翻译影响，因为 证明HO-1通过DAF诱导将C介导的损伤降至最低可能导致新的治疗策略 C依赖型肾脏疾病的治疗干预。

PROJECT SUMMARY / ABSTRACT The glomerular microvasculature is a common target of dysregulated or pathologic complement (C) activation. This has been implicated in the pathogenesis of a wide range of glomerulopathies including lupus nephritis, membranoproliferative glomerulopathy, postinfectious glomerulonephritis and, more recently, the atypical Hemolytic Uremic Syndrome (aHUS), and C3 glomerulopathy. Therefore, development of strategies to minimize activation of C cascades could be promising in C-depended glomerular diseases. The long-term goal of our research is to mitigate severity of C-mediated glomerular injury by up regulating expression of the decay-accelerating factor (DAF), a key controller of C activation acting by preventing assembly and accelerating decay of the naturally labile C3 and C5 convertases that amplify the classical and alternative complement activation cascades. As there are currently no pharmacologic DAF inducers, we sought to identify up regulators of DAF expression whose activity can be increased using existing pharmacologic strategies or agents. We identified Heme Oxygenase (HO)-1, the inducible enzyme of heme degradation to carbon monoxide (CO) and biliverdin, to be a DAF regulator in glomeruli. We demonstrated that HO-1 overexpression targeted to visceral glomerular epithelial cells (also known as podocytes) upregulates DAF and reduces C deposition and extent of injury. Owing to their non-replicative terminally differentiated nature, podocytes are particularly vulnerable to C-mediated injury while their loss was shown to be a key determinant of progression of glomerular diseases. The mechanism by which HO-1 up regulates DAF in podocytes is unknown. Also unknown is the extent to which DAF upregulation by HO-1 is critical in mitigating C-mediated podocyte injury. The proposed studies address these questions by pursuing the following Specific Aims: 1) To identify cis-acting positive response DAF promoter elements in rat podocytes over expressing HO-1, and examine the role of the HO reaction product, CO, as activator of specific DAF promoter transcription factors. This Aim will test the hypothesis that increased CO production in podocytes over expressing HO-1 up regulates DAF expression via CO responsive cis-acting positive regulatory elements on the DAF promoter corresponding to Sp1 transcription factor binding sites. 2) To demonstrate that increased DAF expression is a key mechanism by which HO-1 mitigates complement-dependent podocyte injury. This Aim will test the hypothesis that, in podocytes overexpressing HO-1, DAF mediates attenuation of C-dependent injury The experimental approach in Aim I includes use of cultured podocytes originating from Wild-Type (WT) rats and from rats lacking HO-1 we generated using zinc-finger nuclease (ZFN)-driven HO-1 gene knock- out. In these cells, the effect of HO-1 overexpression and of the HO reaction product, CO, on transcriptional regulation of DAF promoter via the Sp1 transcription factor will be assessed. The experimental approach in Aim II includes use of a rat model of antibody-mediated C-dependent podocyte injury resembling human membranous nephropathy, which will be induced in rats with podocyte- targeted HO-1 overexpression we generated using sleeping beauty (SB) transposon-mediated transgenesis that are either DAF replete or DAF deficient. Observations from the proposed studies are expected to have positive translational impact because the demonstration that HO-1 minimizes C-mediated injury via DAF induction could lead to novel strategies for therapeutic interventions in C-dependent renal diseases.