Clinical Assessment of a novel non-invasive assay for diagnosing recurrent focal and segmental glomerulosclerosis

R03提案的主要目标是开发一种新的基于细胞的检测方法，它将作为一种非侵入性的 一种诊断临床工具，用于检测一种罕见的肾小球疾病，通常称为复发性局灶性肾小球疾病 节段性肾小球硬化(RFSGS)。准确、及时地诊断肾小球疾病是关键 制定成功的治疗计划，从而防止他们进展到终末期肾脏病(ESRD) 疾病)。然而，由于采用了复杂的程序，它们的成功诊断仍然具有挑战性， 包括侵袭性肾活检、免疫组织化学染色和超微结构分析。因此，有一个 迫切需要开发简化的程序来高精度和高精度地检测肾小球疾病患者 效率。重要的是，我们的长期目标是将这一概念扩展到开发其他肾小球的检测方法。 因此，减少或消除了对肾脏活检的需要。尽管有多种原因 对于FSGS，足细胞功能障碍导致细胞死亡和蛋白尿是所有形式的FSGS的主要结果 FSGS。在这项研究中，我们展示了一种专门诊断rFSGS患者的方法的发展。 其中，FSGS在肾移植后数小时至数周内复发，影响超过三分之一的FSGS 患者(因此根据国家稀有疾病组织将其归类为一种罕见疾病)。因此， RFSGS患者的及时诊断将鼓励临床医生执行先发制人的程序，预防 注定要失败的无效的肾移植。由于FSGS的目标是足细胞损伤和死亡，我们独特的 涉及rFSGS患者血浆处理培养的足细胞mRNA谱的方法揭示 上调了与细胞损伤有关的基因。因此，我们选择了三个促凋亡候选基因rFSGS RFSGS患者血浆中特异性升高的反应基因IL1-β、bmf和IGFBP3 足细胞，它们的启动子区域被鉴定并克隆到基于荧光素酶的报告载体和 将其导入Podocytes/HEK293/Cos7细胞，获得稳定的细胞系。令人惊讶的是，当这些细胞系 暴露在rFSGS患者的血浆中，报告活性增加；相反，没有报告活性 在测试的所有其他肾小球疾病患者中都有记录。值得注意的是，统计分析显示 检测rFSGS患者的特异度超过80%。这一概念最近被接受并发表在 顶级肾脏杂志(肾脏国际，在印中)。为了确定这种检测的临床有效性，在 具体目标1，我们将通过测量构建的细胞系的反应来进行临床验证 来自不同肾病患者的血浆。正在招募几名rFSGS和非rFSGS患者 通过各种本土和合作努力为这项研究提供动力。一旦开发出来，这种化验方法将形成 为在世界范围内进行大规模临床试验以充分利用其诊断潜力奠定基础。

The primary goal of this R03 proposal is to develop a novel cell-based assay that will serve as a non-invasive diagnostic clinical tool to detect a rare form of glomerular disease commonly known as recurrent focal and segmental glomerulosclerosis (rFSGS). Diagnosing glomerular diseases accurately and in timely fashion is key to developing a successful treatment plan and thereby prevent their progression to ESRD (end stage renal disease). However, their successful diagnosis remains challenging due to the complex procedures employed, which include, invasive renal biopsy followed by immunostaining and ultrastructural analysis. Thus, there is a compelling need to develop simplified procedures to detect glomerular disease patients with high accuracy and efficiency. Importantly, our long-term goal is to extend this concept to develop assays for other glomerular diseases and therefore, reduce or eliminate the need for renal biopsies. Although there are multiple etiologies for FSGS, dysfunction of podocytes leading to cell death and proteinuria are primary outcomes of all forms of FSGS. In this study, we demonstrate the development of an assay that specifically diagnosed rFSGS patients in which, FSGS recurs following renal transplant within hours to weeks and affects more than a third of FSGS patients (thus classifying it as a rare disorder based on the National Organization of Rare Disorders). Therefore, the timely diagnosis of rFSGS patients will encourage clinicians to execute preemptive procedures and prevent ineffective renal transplants that are destined to fail. Since FSGS targets podocyte damage and death, our unique approach involved mRNA profiling of cultured podocytes treated with rFSGS patient plasma to reveal upregulated genes involved in cellular damage. Therefore,, we selected three proapoptotic candidate rFSGS responsive genes IL1-β, BMF, and IGFBP3 that were specifically elevated in rFSGS patient plasma treated podocytes, their promoter regions were identified and cloned into a luciferase-based reporter vector and transfected into podocytes/HEK293/Cos7 cells to generate stable cell lines. Strikingly, when these cell lines were exposed to plasma from rFSGS patients, increased reporter activity was noted; in contrast, no reporter activity was noted with all the other glomerular disease patients tested. Remarkably, the statistical analysis showed more than 80% specificity in detecting rFSGS patients. This concept was recently accepted for publication in a premier kidney journal (Kidney International, In Press). To determine the clinical effectiveness of this assay, in the Specific Aim 1, we will conduct the clinical validation of constructed cell lines by measuring their responses towards plasma from various nephropathy patients. Several rFSGS and non-rFSGS patients are being recruited through various indigenous and collaborative efforts to power this study. Once developed, this assay will form the basis for conducting worldwide large-scale clinical trials to utilize its full diagnostic potential.