RNA Sequencing of Kidney Immune Cells During Severe AKI Identifies a Potential Role for Granzyme B-Positive CD8 T Cells as a Mediator of Tissue Repair

Background: Various T cells have been implicated in AKI and transition to repair or CKD. However, CD8 T cells have been largely ignored. CD8 T cells have recently been implicated in repair from severe AKI in mice (Kidney Int 2023 PMID: 37011727), however underlying mechanisms are unknown. We performed hypothesis-generating single cell RNA sequencing studies on kidney CD8+ cells during repair from severe AKI. Methods: C57BL6 male mice underwent unilateral renal ischemia reperfusion injury (UIR) with 50-minute ischemia. Kidneys were collected from control and UIR mice after 15 days and CD3+ T cells were flow sorted for 10X Chromium 5’ single cell RNA sequencing (scRNA-seq). Cells were classified using deep learning-based clustering, and differentially expressed genes in each cluster were identified. Gene Set Enrichment Analysis (GSEA) was performed to identify specific genes and pathways that differed between corresponding cell types across conditions. Results: The proportions of CD8 T cells amongst total T cells increased in UIR compared to control (33.9% vs 43.1%, p<0.05). CD8 T cells were classified into 10 clusters, including naïve (L-selectin-positive), granzyme B-positive, and programmed cell death protein 1 (PD1)-positive CD8 T cell clusters. In mice with UIR, granzyme B-positive CD8 T cells increased (6.3% in the UIR vs 0.5% in the control group), and naïve (5.6% in the UIR vs 31.2% in the control group) and PD1-positive CD8 T cells (15.4% in the UIR vs 19.8% in the control group) decreased. Granzyme B-positive CD8 T cells had significantly higher expression of tissue-residency marker genes (Itgae and Itga1), NKT cells marker genes (Klra1, Klra7 and Klrb1c) and genes involved in fibroblast activation (Tgfb1 and Pdgfb) compared to other CD8 clusters. GSEA revealed oxidative phosphorylation (Cox8a, Atp1b1, Cox5a, Cox6c, Cox4i1, Cox5b) and apical surface related genes (Il2rb, Cd160, Flot2, Il2rg) in granzyme B-positive CD8 T cells. Conclusion: These scRNA-seq results demonstrated that a subset of CD8 T cells expressing granzyme B in the kidney during severe AKI is a potential mediator of the switch between repair and progression of AKI to CKD. These data can yield novel mechanisms, biomarkers and therapeutics to enhance repair from severe AKI. Funding: NIDDK Support