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

该R 03提案的主要目标是开发一种新的基于细胞的测定方法，该方法将作为一种非侵入性的 诊断临床工具，以检测一种罕见的肾小球疾病，通常称为复发性局灶性和 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，从而防止他们进展为ESRD（终末期肾功能衰竭） 疾病）。然而，由于所采用的复杂程序，它们的成功诊断仍然具有挑战性， 包括侵入性肾活检，随后进行免疫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，以高准确性检测肾小球疾病患者， 效率重要的是，我们的长期目标是扩展这一概念，以开发其他肾小球疾病的检测方法。 因此，减少或消除对肾活检的需要。虽然有多种病因 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，我们展示了一种特异性诊断rFSGS患者的检测方法的发展。 其中，FSGS在肾移植后数小时至数周内复发，并影响超过三分之一的FSGS 患者（因此根据国家罕见疾病组织将其归类为罕见疾病）。因此，我们认为， rFSGS患者的及时诊断将鼓励临床医生执行先发制人的程序， 注定要失败的无效肾移植由于FSGS靶向足细胞损伤和死亡， 方法涉及用rFSGS患者血浆处理的培养足细胞的mRNA谱分析，以揭示 参与细胞损伤的上调基因。因此，我们选择了三个促凋亡候选rFSGS 在rFSGS患者血浆中特异性升高的IL 1-β、BMF和IGFBP 3应答基因 足细胞，鉴定其启动子区并克隆到基于内切酶的报告载体中， 转染到足细胞/HEK 293/Cos 7细胞中以产生稳定的细胞系。引人注目的是，当这些细胞系 暴露于来自rFSGS患者的血浆，注意到报告子活性增加;相反，没有报告子活性 在所有其他肾小球疾病患者中均观察到。值得注意的是，统计分析显示， 在检测rFSGS患者中特异性超过80%。这一概念最近被接受， 国际肾脏杂志（Kidney International，In Press）为了确定该测定的临床有效性， 具体目标1，我们将通过测量其反应来进行构建细胞系的临床验证 从各种肾病患者的血浆中提取。正在招募几名rFSGS和非rFSGS患者 通过各种本地和合作的努力来推动这项研究。一旦开发出来，这种检测方法将形成 这是进行全球大规模临床试验以充分利用其诊断潜力的基础。

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.