Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury

Abstract

Background: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys.

Methods: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining.

Results: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI.

Conclusion: Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.

Figure 1

Cell lineage analysis in kidney determined by scRNA-seq technology in AKI and control subjects. (A) Fifteen distinct cell populations in kidney were identified by UMAP plot, and each cell was colored representing different subclusters. (B) UMAP plot of cell clusters from distinct subjects with AKI or control, and cells were colored for the individual origin. (C) Bar plot displayed the percentage of cell clusters in kidney from different subjects. Blocks represented distinct subjects, and the cell quantity was in proportion to the block height. (D) Heatmap represented the top 20 most differentially expressed genes of each cell type to identify the cell lineage. Each column reflected an individual cell population, and each row showed marker genes of a single cluster. (E) Violin plot indicated selected marker genes for each cell cluster, and the colors represented marker genes for each cell type. AKI: Acute kidney injury; Con: Control subjects; DC: Dendritic cells; DT: Distal tubule cells; EC: Endothelial cells; FIB: Fibroblasts; scRNA-seq: single-cell RNA sequencing; UMAP: Uniform manifold approximation and projection; SMC: Smooth muscle cells; POD: Podocytes; MES: Mesangial cells; PC: Principal cells; NKT: Natural killer T; IC: Intercalated cells; MON: Monocytes; MC: Macrophages; LOH: Loop of Henle cells; PT: Proximal tubule cell.

Figure 2

DEGs in distinct cell clusters from kidney between ischemic AKI and control subjects. (A) Representative DEGs in glomerulus from AKI patients in comparison with control subjects. (B) Representative DEGs in tubules from AKI patients compared to control subjects. (C, D) Representative DEGs in innate immune cells and adaptive immune cells, respectively, between AKI patients and controls. pct.exp:proportion of cell expressing gene; AKI: Acute kidney injury; DEGs: Differentially expressed genes; IC: Intercalated cells; LOH: Loop of Henle cells; MC: Macrophages; MES: Mesangial cells; MON: Monocytes; NKT: Natural killer T cells; PC: Principal cells; POD: Podocytes; PT: Proximal tubule cells; DC: Dendritic cells; DT: Distal tubule cells; EC: Endothelial cells.

Figure 3

Enrichment analysis in distinct cell clusters from kidney between ischemic AKI and control subjects. (A, C) GO and KEGG enrichment analysis indicated upregulated DEGs involved in biological processes or signaling pathways in different kidney cells. The left side of the circle represented distinct cell clusters, whereas the right side represented distinct biological processes or signaling pathways. The inner circle represented gene numbers enriched in cell clusters or biological processes and signaling pathways. (B, D) The bubble chart exhibited the GO and KEGG enrichment analysis of PT comparing AKI patients to control subjects. count and number of genes annotated to GO terms or KEGG pathways; AKI: Acute kidney injury; DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; IC: Intercalated cells; LOH: Loop of Henle cells; MC: Macrophages; MON: Monocytes; NKT: Natural killer T cells; PC: Principal cells; POD: Podocytes; PT: Proximal tubule cells; DC: Dendritic cells; DT: Distal tubule cells; EC: Endothelial cells.

Figure 4

Ligand–receptor interactions between different cell types in kidney of ischemic AKI patients. (A) Ligand–receptor interactions between different cell types in kidney of AKI subjects, and the frequency of cell–cell communication ranged from low (blue) to high (red). (B–I) Visualized diagrams indicated representative ligand–receptor interactions in MC-MON, MC-EC, MC-MES, MON-DC, MON-DT, MON-EC, EC-MES, and MES-PC, respectively. Lines represented interrelations between the ligand and corresponding receptor. Genes depicted in blue represented ligands, and those displayed in red were receptors. Only AKI patients (n = 2) were analyzed. AKI: Acute kidney injury; MC: Macrophages; MES: Mesangial cells; MON: Monocytes; IC: Intercalated cells; LOH: Loop of Henle cells; PC: Principal cells; POD: Podocytes; PT: Proximal tubule cells; DC: Dendritic cells; DT: Distal tubule cells; EC: Endothelial cells; SMC: Smooth muscle cells; FIB: Fibroblasts.