*Purpose: Studies in our rat model of ischemic reperfusion injury (IRI) demonstrate improved kidney function post injection of non-cultured autologous adipose-derived regenerative cells (ADRC). The mechanism on how these cells induce reparative effects during IRI remains elusive. We investigated ADRC-derived effects on the injured kidney utilizing single cell RNA-sequencing and pathway driven data-mining.

*Methods: ADRC were harvested from inguinal F344 rats (pooled n=5). Stimulated ADRC were exposed for one hour to an ischemic and perfused kidney ex vivo in a transwell system as a mimic of the ADRC treated kidney transplant process and compared to non-stimulated ADRC with no kidney exposure. RNA from single cells was isolated from non-stimulated and stimulated ADRC using the 10X Chromium system and sequencing was performed on a Nextgen 500 at a target 30,000 reads/cell depth (Glasgow Polyomics). Two experimental runs were performed. Post R Seurat sequence analysis, non a priori and directed pathway analysis targeting previous experimental data which implicated anti-inflammatory pathways were evaluated using Ingenuity Pathway Analysis. Sequenced ADRC data results were compared to transcript expression uncovered in the whole rat animal model IRI kidney through qPCR (n=6-8).

*Results: Three merged runs of both Stimulated and Non-stimulated ADRC yielded an average of 6018 cell transcriptomes and 18 distinct cell cluster populations – an enrichment in leukocytes, but also included mesenchymal stem cell and adipocyte populations. Stimulated ADRC yielded 786 cell transcriptomes – primarily T-cell and antigen presenting cell clusters. Cell death transcript expression and low cell yield of treated ADRC suggest higher apoptotic ADRC activity after exposure to the injured kidney. Cell pathways differentially expressed between the two groups implicated a helper T-cell, Th1 profile, an IL-17, and a regulatory pathway, indicating a highly active and proliferating T-cell response. Transcript analysis in IRI and ADRC-treated kidneys showed an increase in cytotoxic T-lymphocyte-associated protein 4 starting at day 48 post treatment. At 48 hours post-treatment, IRI kidneys also contained higher levels of CD45+ leukocytes as assessed by histology and flow cytometry compared to vehicle controls, implicating an immune and regulatory effect of ADRC treatment.

*Conclusions: Pathway analysis in ADRC alone implicate a role for immune cells in modulating the immune response in the IRI kidney. Collectively, gene expression and histological evidence in the model and in ADRC ex vivo suggest that ADRC treated IRI kidneys experience early anti-inflammatory changes involving T-cell and antigen presenting cell subsets that ultimately generate an anti-inflammatory environment in the IRI treated kidney.

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