Introduction: FSGS is a complex pattern of injury that can lead to kidney failure and, consequently, end-stage renal disease. It is well known that podocyte injury and loss is a key pathogenic step. Despite concerted research efforts directed at classifying FSGS using a histologic, genetic, or molecular approach, a detailed understanding of the molecular mechanisms of FSGS pathogenies remain elusive. This study aimed to identify the cellular origin, molecular pathways, and cell-cell interactions contributing to FSGS using a human transplant model.

Methods: Single nuclei RNA-seq was performed on the following human biopsies: i) normal allografts showing non-specific histology (N=4) and ii) FSGS allografts showing glomerular damage (N= 6). Gel-Bead V3 were captured by using the droplet-based 10X Genomic Chromium Platform. Data was analyzed in CellRanger 3.1.0. Downstream analyses were performed including evaluation of cell clusters via uniform manifold approximation and projection, gene and pathway enrichment analyses, intra- and inter-cluster comparative transcriptome analyses.

Results: Using human allografts, a total of 40,078 single nuclei partitioned into 17 unique cell clusters. Stringent quality control metrics were met. We identified both common kidney cell types (e.g. proximal tubular and collecting duct principal cells) and rare cells (e.g. podocytes and immune cells). Comparative analysis between FSGS and normal kidney allografts, revealed a significant decline in endothelial cells and podocytes, as expected. There were 2 novel podocyte subclusters under different transcriptional programs.  The podocyte 1 cluster displayed an injured podocyte phenotype enriched in Wnt signaling and actin filament-based pathways. The podocyte 2 cluster displayed a dysfunctional phenotype enriched in ECM deposition, cell morphogenesis, and cell-cell adhesion pathways. Critically, subcluster analysis revealed 10 endothelial cell types, which included glomerular, peritubular capillary, and arteriolar cells, and 6 immune cell clusters, which included macrophages/ monocytes, T cells, natural killer cells, and B cells. Immune cells were significantly increased, specifically in T memory cells, and their transcriptional profile was significantly altered in FSGS patients compared to normal.

Conclusions: FSGS is characterized by a complex cellular and transcriptomic landscape, leading to further kidney injury.