Single-nuclear transcriptomics reveals diversity of proximal tubule cell states in a dynamic response to acute kidney injury

Abstract

Acute kidney injury (AKI), commonly caused by ischemia, sepsis, or nephrotoxic insult, is associated with increased mortality and a heightened risk of chronic kidney disease (CKD). AKI results in the dysfunction or death of proximal tubule cells (PTCs), triggering a poorly understood autologous cellular repair program. Defective repair associates with a long-term transition to CKD. We performed a mild-to-moderate ischemia-reperfusion injury (IRI) to model injury responses reflective of kidney injury in a variety of clinical settings, including kidney transplant surgery. Single-nucleus RNA sequencing of genetically labeled injured PTCs at 7-d ("early") and 28-d ("late") time points post-IRI identified specific gene and pathway activity in the injury-repair transition. In particular, we identified Vcam1⁺/Ccl2⁺ PTCs at a late injury stage distinguished by marked activation of NF-κB-, TNF-, and AP-1-signaling pathways. This population of PTCs showed features of a senescence-associated secretory phenotype but did not exhibit G₂/M cell cycle arrest, distinct from other reports of maladaptive PTCs following kidney injury. Fate-mapping experiments identified spatially and temporally distinct origins for these cells. At the cortico-medullary boundary (CMB), where injury initiates, the majority of Vcam1⁺/Ccl2⁺ PTCs arose from early replicating PTCs. In contrast, in cortical regions, only a subset of Vcam1⁺/Ccl2⁺ PTCs could be traced to early repairing cells, suggesting late-arising sites of secondary PTC injury. Together, these data indicate even moderate IRI is associated with a lasting injury, which spreads from the CMB to cortical regions. Remaining failed-repair PTCs are likely triggers for chronic disease progression.

Keywords: acute kidney injury; proximal tubule; repair; single-nucleus RNA sequencing; transcriptomics.

Fig. 1.

Krt20 inducible Cre-LoxP system traces injured PTCs in post-IRI kidneys. (A) Schematic diagram of CRE-ERT2 (CE) knocked into Krt20 genomic locus through CRISPR. (B) Experimental setup. (C) Colocalization of GFP reporter with Krt20 and Havcr1 on IRI samples. (Scale bar, 20 μm.) (D) Quantification of GFP⁺ cells per region of interest (ROI) using five ROIs per kidney section (n = 2). (E) Quantification of GFP⁺ cells per ROI in cortex and cortico-medullary boundary (CMB) using 10 cortical and CMB ROIs per kidney section (n = 2). (F) Quantification of colocalization of GFP reporter, Krt20, and Havcr1 per ROI, 10 ROIs per kidney section (n = 2). (G) UMAP plot of the integrated IRI and control single-nuclei RNA-sequencing datasets. (H) Violin plots of marker genes arranged by cell type; cluster numbers are indicated above the plots. In D–F, the gray box corresponds to the middle 50th percentile, the horizontal line to the median, and the whiskers indicate the 1.5 interquartile range. ***P < 0.001.

Fig. 2.

PTCs adopt transcriptionally diverse cell states in response to injury. (A) UMAP plot of the PTC clustering. (B) Dot plot of cluster enriched gene expression. Violin plot of male-specific S3 segment marker Cyp7b1 and female-specific S3 segment marker Slc7a12 across clusters split in IRI and control cells. (C) Stacked bar plot of the composition of clusters by group. (D) GO analysis of biological processes and cellular components (F) across clusters. Top three enriched GO terms per cluster are shown. (E) Gene-regulatory network analysis using single-cell regulatory network inference and clustering (SCENIC) across IRI cells. Heatmap showing regulon activity across clusters. Transcription factors representative for each group are highlighted. See Dataset S3 for a complete list of identified regulons.

Fig. 3.

Validation of identified injured PTC states. (A) A combination of RNAscope (Slc22a7 and Slc7a12) and immunofluorescence (Vcam1 and Aqp1) in the same sections shows up-regulation of female-specific gene Slc7a12 in male mice after IRI. (B–D) Immunofluorescence staining of Cdh6, Cdh13, Ki67, Vcam1, Hnf4a, and Lrp2 in proximal tubules validates identified PTC states. (Scale bar: A–D, 20 μm.)

Fig. 4.

Vcam1⁺/Ccl2⁺ proximal tubule subpopulation shows proinflammatory and profibrotic signature, but no G₂/M cell cycle arrest. (A) Top 20 enriched KEGG pathways in late injured cluster 11. (B) Depiction of the KEGG pathways TNF and NF-κB signaling with the involved genes up-regulated in cluster 11. (C) Violin plots displaying expression of selected features across proximal tubule clusters. The cluster numbers correspond to Fig. 2A. (D) Gene set enrichment analysis of published cell cycle and (E) senescence/senescence-associated secretory phenotype (SASP) gene sets across clusters. See Dataset S4 for details on used gene sets.

Fig. 5.

Immunofluorescence and RNAscope validate Nfkb1 expression in Vcam1⁺/Ccl2⁺ PTCs. (A) Immunofluorescence staining validates Nfkb1 and Vcam1 in Krt20⁺ cells. (B) Combination of RNAscope in situ hybridization (Nfkb1, Ccl2, GFP) and immunofluorescence staining (Vcam1) shows Nfkb1, Ccl2, Vcam1, and Krt20-INTACT GFP-coexpressing cells. (C and D) Quantification of (C) colocalization of Nfkb1 with Ccl2 in PTCs and of (D) Ccl2, Vcam1 and Nfkb1 in GFP⁺ PTCs using five regions of interest (ROIs) per kidney section (n = 3). The gray box corresponds to the middle 50th percentile, the horizontal line to the median, and the whiskers indicate the 1.5 interquartile range. (E) Combination of RNAscope in situ hybridization (Il34, Dock10) and immunofluorescence staining (Vcam1, Krt20-TDT) shows Il34 and Dock10 expression in Vcam1⁺, Krt20⁺ cells marked by the fluorescent reporter tandem tomato (TDT) in Krt20^T2A-CE/+;R26R^tdTomato/+ mice. (F) Immunofluorescence staining shows Cd45⁺, Nfkb1⁺ inflammatory cells around the Nfkb1⁺ tubules. See SI Appendix, Fig. S4E for validation of Nfkb1 antibody. (G) Box plots of reads per kilobase per million mapped reads (RPKM) values of indicated genes in bulk RNA-seq analysis of human kidney transplants biopsies in a successfully repaired [1] or a transition state [2] toward a chronically injured state [3]. Biopsy samples from living donors (LDs) were included as a representation of normal kidney tissue. Groups were compared by Mann–Whitney U test. Box corresponds to the middle 50th percentile, the horizontal line to the median, and the whiskers indicate the data range. Data adopted from ref. . (Scale bar: A, B, E, F, 20 μm.)

Fig. 6.

The temporal and spatial relationship of PTC injury signatures in the mouse kidney. Lineage tracing of proliferating cells using a Ki67^Cre/ERT2;INTACT mouse line with tamoxifen (Tam.) injections either on day 2 and 3 or on day 5 and 6 post-IRI. (A) RNAscope in situ hybridization (Ccl2, Krt20, GFP) combined with immunofluorescence staining (Vcam1) shows colocalization of Ki67-INTACT GFP-positive cells with Vcam1, Krt20, and Ccl2 on day 28 post-IRI when tamoxifen is injected on day 2 and 3 post-IRI. (B) Quantification of GFP reporter in Ccl2⁺/Vcam1⁺ cells in the cortex and the cortico-medullary boundary (CMB) at day 28 post-IRI in mice injected with tamoxifen either on day 2 and 3 (n = 4) or on day 5 and 6 (n = 3) post-IRI. (C) Immunofluorescence staining of proximal tubules with Krt20, Vcam1, Hnf4a, and LTL and quantification (D) of Vcam1⁺ PTCs in uninjured kidneys of 3- and 18-mo-old C57BL/6N mice. The gray box corresponds to the middle 50th percentile, the horizontal line to the median, and the whiskers indicate the 1.5 interquartile range. *P < 0.05. (Scale bar: A and C, 20 μm.)