Cytokine based murine focal glomerulosclerosis model a prelude to novel therapy

描述（由申请人提供）： 总结：局灶节段性肾小球硬化（FSGS）是慢性肾功能衰竭最常见的原发性肾小球原因，需要透析和移植。大约1/3的FSGS患者在移植后迅速复发蛋白尿。复发似乎取决于血浆“因子”的存在，该因子损伤同种异体移植物的肾小球。我们和其他研究者已经证明，移植后复发性FSGS患者的血浆增加了肾小球白蛋白的渗透性，并导致实验动物的蛋白尿。对于这种形式的FSGS，没有稳健的动物模型。有害物质尚未被鉴定并且没有适合于测试潜在疗法的动物模型的事实限制了对血浆因子诱导的FSGS（在本申请中称为piFSGS）的治疗的研究。因此，治疗试验仍然是经验性的，必须专门在受影响的患者中进行。我们的长期目标是开发特异性治疗以逆转早期piFSGS中的蛋白尿，阻止肾损伤的进展，并预防piFSGS的移植后复发和随后的同种异体移植物损失。我们已经使用最先进的蛋白质组学技术，以确定细胞因子，心肌营养素样细胞因子-1（CLC-1）作为该血浆中的活性成分。我们建议建立一个小鼠模型的piFSGS的基础上，这种细胞因子的影响。该模型将提供一个强大的工具来定义肾脏对CLC-1的反应和测试候选治疗。该模型的成功开发和使用将允许为人类FSGS的新疗法的未来试验设计策略。我们将讨论三个主要假设。第一：慢性升高的CLC-1血浆浓度导致蛋白尿和肾损伤。第二：针对CLC-1下游效应的治疗将预防蛋白尿和肾损伤的进展。第三：针对由CLC-1触发的细胞信号传导的治疗将预防蛋白尿和肾损伤的进展。具体目的是：1：使用CLC-1输注或表达建立人血浆诱导的FSGS（piFSGS）的鼠模型。将CLC-1输注，通过转染含有CLC-1基因的质粒过表达CLC-1或在转基因小鼠的肾外组织中表达。读数将包括蛋白尿、肾组织学、足细胞超微结构和足细胞特异性基因的表达改变，以及2：测试CLC-1诱导的肾病的潜在治疗剂。将测试3类代理。这些药物包括特异性结合FSGS因子和/或CLC-1并阻止其对肾小球的作用的药物，可以通过作用于肾小球本身来保护滤过屏障的药物，以及已经显示出作为人类抗蛋白尿和抗纤维化药物的前景的其他药理学药物。将通过预防蛋白尿和足细胞改变、逆转已建立的肾小球损伤、阻止肾瘢痕形成进展和已建立病变愈合来评估有效性。3：定义对CLC-1的早期足细胞应答，以鉴定在piFSGS中早期干预的潜在靶标。将使用用CLC-1孵育的培养的永生化鼠足细胞（Mundel）进行研究。我们将探索通过JAK/STAT，PI 3 K/AKT和MAPK/ERK途径的信号转导的作用，使用Western印迹法，有或没有事先免疫沉淀。我们将使用药理学抑制剂，当可用时，以确认信号通路之间的关系。这些初步研究将允许设计未来更详细和具体的研究CLC-1对足细胞的影响。总之，这些建立和表征piFSGS鼠模型的研究将与移植后肾病快速进展和/或复发的FSGS患者独特相关。该方法结合了经验疗法和基本细胞信号传导反应的研究，将允许设计未来用于人类的有效疗法。

DESCRIPTION (provided by applicant): Summary: Focal segmental glomerulosclerosis (FSGS) is the most common primary glomerular cause of chronic renal failure requiring dialysis and transplantation. Proteinuria recurs promptly after transplantation in about 1/3 of patients with FSGS. Recurrence appears to depend on the presence of a plasma "factor" that injures the glomeruli of the allograft. We and other investigators have shown that plasma of patients with recurrent FSGS post-transplant increases glomerular albumin permeability and causes proteinuria in experimental animals. There is no robust animal model for this form of FSGS. The facts that the injurious substance has not been identified and that there is no animal model suitable for testing potential therapy have limited studies treatment for plasma-factor-induced FSGS (termed piFSGS in this application). Thus, trials of therapy remain empiric and must be carried out exclusively in affected patients. Our long term goal is to develop specific therapy to reverse proteinuria in early piFSGS, to arrest progression of renal injury and to prevent post-transplant recurrence of piFSGS and consequent allograft loss. We have used state-of-the-art proteomics techniques to identify a cytokine, cardiotrophin-like cytokine-1 (CLC-1) as an active component in this plasma. We propose to establish a murine model of piFSGS based on the effects of this cytokine. This model will provide a powerful tool to define the renal responses to CLC-1 and test candidate therapy. Successful development and use of this model will permit design of strategies for future trials of novel therapies in human FSGS. We will address 3 major hypotheses. First: Chronically elevated plasma concentration of CLC-1 results in proteinuria and renal injury. Second: Therapies directed at the downstream effects of CLC-1 will prevent proteinuria and progression of renal injury. Third: Therapies directed at cell signaling triggered by CLC-1 will prevent proteinuria and progression of renal injury. Specific Aims are: 1: To create a murine model of human plasma-induced FSGS (piFSGS) using CLC-1 infusion or expression. CLC-1 will be infused, overexpressed CLC-1 by transfection of plasmids containing the CLC-1 gene or expressed in extrarenal tissues of transgenic mice. Read-outs will include proteinuria, renal histology, podocyte ultrastructure and altered expression of podocyte-specific genes, and 2: To test potential therapeutic agents in CLC-1 induced renal disease. Agents in 3 categories will be tested. These include agents that specifically bind to the FSGS factor and/or CLC-1 and prevent its effect on the glomerulus, agents that may protect the filtration barrier through action on the glomerulus itself and additional pharmacological agents that have shown promise as antiproteinuric and antifibrotic agents in humans. Effectiveness will be assessed by prevention of proteinuria and podocyte alterations, reversal established glomerular injury, arrest of progression of renal scarring and healing of established lesions. 3: To define early podocyte responses to CLC-1 in order to identify potential targets of early intervention in piFSGS. Studies will be conducted using cultured immortalized murine podocytes (Mundel) incubated with CLC-1. We will explore the role of signaling via JAK/STAT, PI3K/AKT and MAPK/ERK pathways using Western blotting with or without prior immunoprecipitation. We will use pharmacological inhibitors, when available, to confirm relationships among signaling pathways. These preliminary studies will permit design of future more detailed and specific studies CLC-1 effects on podocytes. Taken together these studies to establish and characterize a murine model of piFSGS will be uniquely relevant to FSGS patients with rapid progression of renal disease and/or recurrence after transplantation. The approach, which combines studies of empiric therapies and of basic cellular signaling responses, will permit the design of effective therapy for future use in humans.