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-?, 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.
This study uses a novel cell-based approach to develop diagnostic kit for detecting a rare form of glomerular disease known as recurrent focal and segmental glomerulosclerosis (rFSGS). This assay will be highly significant in identifying FSGS patients that are destined to loose renal graft following renal transplant. Importantly, this revolutionary approach can widely be used in developing similar assays for several other rare glomerular diseases.
