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

Abstract

BackgroundCurrent 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.MethodsWe 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.ResultsOur 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.ConclusionsThe 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.

The dysregulation of metabolic pathways and induction of the pentose phosphate pathway in renal ischaemia–reperfusion injury

“…Consistent with the results obtained in the in silico analysis, the expression of PPP‐related genes – G6pdx , Taldo1 , Tkt , and Prps1 – was significantly increased in ischaemic mice, compared with sham mice (Figure 6E–H). It is important to note that elevated expression of PPP‐related genes was also observed during the repair phase in a publicly available single cell RNA dataset [29] (supplementary material, Figure S7E–H).…”

“…Our data imply that during the reperfusion phase, the cell strategically chooses the PPP over glycolysis. Once metabolic functions have been restored, it does not seem advantageous for the cell to shuttle glucose through a pathway that does not yield ATP; however, our analysis of a single cell RNA dataset [29] showed that increased expression of several PPP‐related genes is maintained for up to 7 days following IRI in the proximal tubular cell population. This further suggests that the activation of the PPP may be responsible for the generation of ‘chemical supplies’ needed for cellular repair.…”

A Partial Picture of the Single-Cell Transcriptomics of Human IgA Nephropathy

“…The fast development of single-cell RNA sequencing (scRNA-seq) technology allows the inquiry of transcriptomic profiles and signaling pathways in diverse cell types from a given sample simultaneously, and unlike bulk RNA-seq, it can define comprehensive gene sets at the single-cell level (9,10). Recently, this new methodology has been employed in various renal diseases (11)(12)(13)(14). In the present study, we applied scRNAseq to kidney samples from patients with IgAN to identify gene expression at the single cell level, and explore novel cellular interactions and crucial molecular pathways contributing to the disease development.…”

Molecular Mechanisms of Renal Progenitor Regulation: How Many Pieces in the Puzzle?

“…This novel technology has been used successfully in several organs—for example, to study Prominin 1+ liver progenitors [ 114 ], Dach1–downregulated lymphoid progenitors [ 115 ] and KTR5+ lung progenitors in COVID-19 patients [ 116 ]. Within the past few years, an increasing number of research groups have applied this strategy to define the cell populations of the kidneys in mice and humans [ 117 , 118 , 119 , 120 ]. In a very recent study, Rudman-Melnick et al identified the transcriptional signature of all cell populations in an experimental model of AKI, highlighting the presence of previously undescribed injury-related molecules [ 119 ].…”

“…Within the past few years, an increasing number of research groups have applied this strategy to define the cell populations of the kidneys in mice and humans [ 117 , 118 , 119 , 120 ]. In a very recent study, Rudman-Melnick et al identified the transcriptional signature of all cell populations in an experimental model of AKI, highlighting the presence of previously undescribed injury-related molecules [ 119 ]. Such an approach could potentially reveal novel mechanisms activated in renal progenitors following AKI, leading to the identification of potential molecular targets.…”