*Purpose: Focal segmental glomerulosclerosis (FSGS) remains a disease without specific therapy for primary disease and a high rate of recurrence after renal transplant. Circulating factors are implicated in the pathogenesis of FSGS but targeting them for therapy has remained elusive. We have previously described a panel of autoantibodies in sera from FSGS patients to predict the risk of recurrence after transplant with high accuracy. Furthermore, we showed interaction between two circulating factors- soluble urokinase-like plasminogen activator receptor (suPAR) and autoantibodies to CD40 in augmenting podocyte injury in vitro and in vivo. These studies suggested that anti uPAR and anti CD40 therapies may be effective in slowing down or reversing the renal injury leading to recurrence after transplant. The study presented here was conducted with two goals: 1) Validate the of role of CD40 and suPAR pathways in direct podocyte injury, and 2) Identify the molecules and pathways uniquely associated with CD40 and suPAR to find new markers and therapeutic candidates associated with podocyte injury in FSGS.

*Methods: Sera from three patients with biopsy proven FSGS and recurrence of FSGS after transplant was used to cause injury in immortalized human podocytes in culture. The efficacy of an anti uPAR antibody, 2G10 (developed by Dr. Charles Craik and described previously) or anti CD40 antibody (BMS 986090) was tested to reverse podocyte injury. For global transcriptomic profiling RNA was isolated from podocytes treated with anti CD40 antibody purified from patients with recurrence of FSGS, suPAR or both and subjected to whole Human Genome Microarray 4X44K (Agilent Technologies) for gene expression analysis.

*Results: Podocyte culture in the presence of sera from all three patients caused significant depolarization of stress fibers as determined by number of stress fiber positive cells (30%, 59% and 49% reduction with respected to untreated podocytes respectively), a hallmark of podocyte injury. Culture of podocytes with patient sera in the presence of anti CD40 antibody or 2G10 antibody (1ug/mL) against uPAR rescued stress fibers. CD40Ab caused upregulation of inflammatory genes involved in the regulation of type I interferon signaling pathway, cytokine-mediated signaling pathway and endothelial cell migration. Combined administration of patient derived CD40Ab and suPAR resulted in a primary response to suPAR with activation of genes involved in megakaryocyte differentiation. However, the addition of CD40Ab caused additional changes that are associated with integrin signaling.

*Conclusions: This study provides a proof of concept that inhibition of uPAR and CD40 signaling in podocytes can be an effective treatment strategy for recurrence of FSGS and potential abrogation of FSGS disease and renal allograft salvage.

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