Cytokine based murine focal glomerulosclerosis model a prelude to novel therapy

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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.