Single-Cell Profiling of AKI in a Murine Model Reveals Novel Transcriptional Signatures, Profibrotic Phenotype, and Epithelial-to-Stromal Crosstalk

Significance Statement Because current management of the rapid renal-function decline in AKI is merely supportive, deeper understanding of the AKI-perturbed molecular pathways is needed to identify targets with potential to lead to improved treatment. In a murine AKI model, the authors used single-cell RNA sequencing, single-molecule in situ hybridization, and protein expression analyses to create the first comprehensive renal cell type–specific transcriptional profiles for multiple AKI stages. Their findings revealed a marked nephrogenic signature and surprising mixed-identity cells (expressing markers of different cell types) in the injured renal tubules. Moreover, the authors identified potential pathologic epithelial-to-stromal crosstalk and several novel genes not previously implicated in AKI, and demonstrated that older onset age exacerbates the AKI outcome. This work provides a rich resource for examining the molecular genetics of AKI. Background Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment. Methods We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments. Results Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors—including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)—and of several novel genes ( Ahnak , Sh3bgrl3 , and Col18a1 ) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a . Moreover, AKI caused the formation of “mixed-identity cells” (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis. Conclusions The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.