*Purpose: Focal Segmental Glomerulosclerosis (FSGS) is the most common cause of nephrotic syndrome, can lead to ESRD, and carries a high burden of recurrence risk an accelerated graft loss after transplant, with associative circulating factors with suggested pathogenesis. We hypothesized that FSGS may be a disease caused by dysregulated hematopoietic stem cells that result in the production and release of circulating factors which result in podocytopathy.

*Methods: Iliac crest bone marrow and sera was obtained with informed consent from patients with primary FSGS as their cause of ESRD with either lack of recurrence (nrFSGS; n=4), loss of a first renal allograft due to biopsy confirmed FSGS recurrence (rFSGS; n=4), ESRD due to causes other than FSGS (nFSGS; n=2), or without ESRD as healthy controls (HC; n=2). Bone marrow was sorted into either unfractionated bone marrow cells (BMC) or into flow sorted mononuclear cells (BM-MNC). In vitro culture of BMC allowed for characterization of specific soluble factors which were subsequently tested on a custom immortalized human podocyte cell line and for their ability to cause podocyte injury. Multiplex single-cell RNASeq (Mux-Seq) of BMC and BM-MNC was conducted on all samples with SNP based deconvolution of individual samples, using NovaSeq. Data analysis for podocyte injury was done by confocal microscopy and actin depolarization MFI and for Mux-Seq by SEURAT, with scRNASeq data visualization by tSNE/uMAPs.

*Results: Sera from rFSGS but not nrFSGS patients causes actin cytoskeleton rearrangement and loss of stress fibers as a hallmark of podocyte injury, confirming the presence of one/more systemic soluble podocytopathic factors. Treatment with conditioned media from rFSGS BMC culture but not from healthy control BMCs also resulted in podocyte damage. Mux-Seq of BMC and BM-MNC revealed at least18 distinct cell populations, with subsets of unique MNC populations in rFSGS, nrFSGS, nFSGS and healthy BM, with differential expression of specific proteins mapped to previous autoantibodies studied in rFSGS, inclusive of PLAUR and CD40.

*Conclusions: Podocyte injury in rFSGS is supported by one or more circulating autoantibodies or proteins, but further work is needed to dissect causation from association. Knock out animal studies are underway to address this. Mux-Seq provides tantalizing results on unique MNC subsets in rFSGS with variations in specific proteins relating to the uPAR/CD40 pathway that are currently undergoing further investigation.

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