Novel Complement-targeted treatment strategies in Renal Disease

肾病补体靶向治疗新策略

• 批准号: 10057225
• 负责人: Elias Lianos
• 金额: --
• 依托单位: SALEM VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057225
• 关键词: Address; Animal Model; Antibodies; Basic Science; Biliverdine; Binding Sites; CD55 Antigens; CRISPR/Cas technology; Carbon Monoxide; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; Complement 3 Convertase; Complement Activation; Complement Inactivators; Deposition; Development; Disease; Elements; Enzymes; Exons; Exposure to; Gene Transfer Techniques; Genes; Genomics; Glomerulonephritis; Goals; Heme; Hemolytic-Uremic Syndrome; Host Defense; Human; Immunity; Injury; Injury to Kidney; Kidney Diseases; Knock-out; Lead; Lupus Nephritis; Malignant neoplasm of prostate; Mediating; Medical; Membranous Glomerulonephritis; Messenger RNA; Microbe; Modeling; Mus; Nature; Organ; Oxygenases; Pathogenesis; Pathologic; Pharmacology; Physiological; Production; Promoter Regions; Proteins; Rattus; Reaction; Regulation; Regulatory Element; Renal glomerular disease; Research; Research Proposals; Response Elements; Role; Severities; Sleeping Beauty; Sp1 Transcription Factor; System; Testing; Therapeutic Intervention; Transcriptional Regulation; Transgenic Organisms; Up-Regulation; Veterans; attenuation; base; heme oxygenase-1; innovation; interest; kidney cell; malignant breast neoplasm; novel; novel therapeutic intervention; overexpression; podocyte; prevent; promoter; protein activation; response; targeted treatment; therapeutic target; transcription factor; translational impact; treatment strategy; zinc finger nuclease

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.

项目总结/摘要 肾小球微血管是失调或病理性补体的常见靶点（C） activation.这与包括狼疮在内的多种肾小球疾病的发病机制有关 肾炎，膜增生性肾小球病，感染后肾小球肾炎，以及最近， 非典型溶血性尿毒综合征（阿胡斯）和C3肾小球病。因此，制定战略， 最小化C级联的激活在C依赖性肾小球疾病中可能是有希望的。 我们研究的长期目标是减轻C介导的肾小球损伤的严重程度， 调节衰变加速因子（decay-accelerating factor，简称ERF）的表达，ERF是C激活的关键控制因子， 阻止组装并加速扩增所述细胞的天然不稳定的C3和C5转化酶的衰变， 经典和替代补体激活级联。由于目前还没有药理学上的 诱导剂，我们试图确定上调调节剂的表达，其活性可以增加使用现有的 药理学策略或药剂。我们鉴定了血红素加氧酶（HO）-1， 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 针对内脏肾小球上皮细胞（也称为足细胞）的HO-1过表达上调 减少C沉积和损伤程度。由于它们的非复制性终末分化 自然界中，足细胞特别容易受到C介导的损伤，而它们的损失被证明是一个关键因素。 肾小球疾病进展的决定因素。 HO-1在足细胞中上调IL-6的机制尚不清楚。同样未知的是 其中HO-1的上调对减轻C介导的足细胞损伤至关重要。拟议的研究 通过追求以下具体目标来解决这些问题： 1)为了在大鼠足细胞中鉴定顺式作用的阳性反应启动子元件， 表达HO-1，并检查HO反应产物CO作为特异性转录因子的激活剂的作用。 启动子转录因子该目的将检验足细胞中CO产生增加的假设， 过表达HO-1通过CO反应性顺式作用正调控元件上调TGF β 1表达 在对应于Sp1转录因子结合位点的P2P启动子上。 2)为了证明增加的H2 O2表达是HO-1减轻H2 O2表达的关键机制， 补体依赖性足细胞损伤。这一目的将检验这一假设，即在足细胞过度表达 HO-1，HO-2介导C依赖性损伤的减轻 目的I中的实验方法包括使用来源于野生型小鼠的培养足细胞。 (WT)大鼠和缺乏HO-1的大鼠，我们使用锌指核酸酶（ZFN）驱动的HO-1基因敲除产生， 出去在这些细胞中，HO-1过表达和HO反应产物CO对转录的影响， 将评估通过Sp1转录因子对P2P启动子的调节。 目的II中的实验方法包括使用抗体介导的C-依赖性 足细胞损伤类似于人类膜性肾病，这将在足细胞- 我们使用睡美人转座子介导的转基因技术， 要么是营养过剩要么是营养不足。 预计拟议研究的观察结果将产生积极的翻译影响，因为 证明HO-1通过β诱导最大限度地减少C介导的损伤可能导致新的策略， C依赖性肾病的治疗干预。