Objectives/Goals: The identification of the cascade of molecular and cellular events occurring during the progression of focal segmental glomerulosclerosis in human kidney biopsies from kidney transplant (KTx) recipients (KTR) with normal function or recurrent FSGS to determine potential targets of intervention and therapy. Methods/Study Population: In this study, we evaluate the molecular and cellular events associated with primary FSGS in both native and transplant kidneys. We collected biopsy samples from the native normal kidney (nNK, n = 3), normal functioning allografts (NKTx, n = 3), primary FSGS in the native kidney (nFSGS, n = 1), recurrent FSGS (KTxFSGS, n = 5). KTxFSGS comprises a collection of longitudinal samples with biopsy also collected at the subsequent recurrence. Blood samples were collected during biopsy collection. Biopsies were preserved in RNAlater at the time of collection. 10X genomics chromium single nuclei RNA sequencing (snRNAseq) was performed using isolated nuclei. Data was analyzed using Seurat on R. Conditionally immortalized podocytes were treated with a patient serum to determine the change in expression observed in snRNAseq data. Results/Anticipated Results: Recurrence rates of primary FSGS are high in kidney allograft recipients up to 25–50% in first, and up to 80% in second transplants, often leading to graft loss. Our findings reveal that podocyte detachment is driven by metabolic and structural dysregulation rather than cell death, increasing VEGFA expression and disrupting glomerular endothelial cell growth and permeability. Parietal epithelial cells initially compensate by dedifferentiating toward podocytes but later increase collagen deposition, contributing to glomerular sclerosis. Increased interactions of glomerular cells with B cells exacerbate extracellular matrix deposition and scarring. We also observed tubular sclerosis and disruption of the regenerative potential of proximal tubular cells, with increased interaction with T cells. Discussion/Significance of Impact: These findings offer new insights into the pathogenesis of recurrent FSGS and suggest potential therapeutic targets and establishes a foundation for future studies to further evaluate the role of metabolic dysfunction as the cause of podocyte injury and loss.