Single-Cell RNA Sequencing of Kidney-Gut Axis during Microbiome Manipulation of Severe AKI to CKD Progression

Background: The gut microbiome modifies experimental acute kidney injury (AKI) outcomes. The antibiotic drug amoxicillin, accelerates kidney repair and decreases fibrosis after severe AKI, likely through CD8 T cells (Kidney International, 2023). The goal of this study was to uncover specific genes and pathways that mediate T cell response to amoxicillin and regulate recovery and repair from severe AKI. Methods: 8-10 week old male C57BL6 mice underwent unilateral ischemia reperfusion injury (UIRI) to kidney (n=10/group). Mice were given vehicle (1% glucose) or amoxicillin (50 mg/ml) in drinking water starting soon after UIRI and kidney and colon tissues collected after 15 days. Non surgery mice treated with vehicle and amoxicillin were used as controls. CD3+ cells were flow sorted for 10X Chromium 5-prime single cell RNA sequencing (scRNA-seq). Cells were annotated using machine learning classification and manual annotation using established cell markers. Differential gene expression and Gene Set Enrichment Analysis (GSEA) was performed to identify specific genes and pathways that differed between corresponding cell types across conditions. Results: After quality control, we recovered 22,260 cells from the kidney and 14,669 cells from the colon. Nine cell types were annotated, including Th1, Th2, Th17, regulatory T cells, effector and memory CD8+ cells, and naive CD4+, CD8+, double negative and double positive T cells. Amoxicillin treatment after UIRI increased the proportion of CD8+ T cells and reduced that of CD4+ T cells in kidney, while the colon decreased the fraction of CD8+ T cells. GSEA revealed aldosterone-regulated sodium reabsorption (downregulated Prkcb, Insr, Nedd4l, etc) and JAK-STAT signaling pathway in kidney (downregulated Jak1, Ccnd3, and Socs1, etc), as well as estrogen signaling pathway (downregulation was repaired after Amoxicillin, marked by Jun, Fos, Hsp90aa1, etc) and renin secretion (downregulated Gnai2, Adrb1, Gnas, etc) in the colon. Conclusion: These scRNA-seq results show shifts in T cell populations in gut-kidney axis during severe AKI and corroborated our prior flow cytometry observations. Our scRNA-seq data and analyses promise to yield novel mechanisms, markers, therapeutic options for severe AKI and gut microbiome-kidney cross talk. Funding: NIDDK Support