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  1. Reviewer #3 (Public Review):

    In this study, Ide et al present a comprehensive analysis of single cell transcriptomic changes in the kidney in response to mild and recoverable injury compared to severe and persistent injury after renal ischemia reperfusion in an effort to identify cellular pathways that promote maladaptive repair. The analysis of their transcriptomic data identify pathways that persist in severe injury, confirming findings identified by other groups including induction of cell populations involved in the repair response after injury (SOX9-expressing cells), cellular interactions (tubular-derived chemokines/cytokines to monocyte/macrophage receptors), and cellular pathways (Gpx4-glutathione) that are important to prevent ferroptosis, a major pathway known to drive cell death in renal ischemia-reperfusion injury. Global deletion Gpx4 has been shown by others to drive ferroptotic cell death in renal ischemia-reperfusion. Ide et al use a genetic model of Sox9-specific deletion of Gpx4 to show that deletion of Gpx4 specifically in Sox9-expressing cells is sufficient to enhance ferroptotic cell death and maladaptive repair.

    Strengths:

    The strengths of this manuscript include the generation of a large dataset of scRNA-Seq in mild versus severe ischemic kidney injury at several time points as well as validation with qPCR and immunostaining of a subset of pathways and cellular injury markers identified in their scRNA-Seq analyses. Cellular differentiation and developmental pathways activated in the course of injury and repair in the adult kidney were also analyzed in relation to publicly available neonatal kidney scRNA-Seq data. Additional correlation of identified pathways and cellular injury markers were further supported by analyses of previously published scRNA-Seq data from human kidney biopsy samples from normal controls and non-rejection acute kidney injury allografts.

    Weaknesses:

    Data presented in the manuscript confirms previously published known findings and does not present a novel observation or pathway. Additional discussion should be included to acknowledge and explain the assumptions and limitations of the scRNA-Seq analyses, particularly the RNA velocity and pseudo time trajectory analyses, which are mathematical models based on assumptions of gene expression similarity to impute cellular transition states.

    The conclusions of this paper are mostly well supported by data, but some aspects of the immunostaining need to be clarified. In particular the variability of VCAM1 staining across figures especially in the control contralateral kidney and the difference between SOX9 immunostaining as compared to the Sox9-TdTomato reporter.

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  2. Reviewer #2 (Public Review):

    In this work, the authors addressed whether different times of ischemia in kidney may have a different outcome on the regenerative capacity of kidney tubular cells. The authors applied mild and severe ischemia to kidney tissue and performed scRNA sequencing to identify RNA signatures and trajectories that are predictive whether or not cells may activate a regenerative program that allows to repair damaged tissues. The findings obtained from the animal studies were then compared with human kidney samples where similar signatures could be detected. The authors went on and generated an elegant conditional mouse model with ischemic stress-induced inactivation of the key ferroptosis regulator glutathione peroxidase 4 (GPX4). This model is based on the mild stress-induced upregulation of endogenous Sox9 driving Cre expression and thus deletion of GPX4. In general, this is an easy to follow and intriguing study with many new exciting insights that should help in understanding the role of ferroptosis in the development of ischemia/reperfusion injury in kidney disease.

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  3. Reviewer #1 (Public Review):

    Ide and colleagues report on an undescribed, pro-inflammatory proximal tubule cell state (DA-PT) in the pathogenesis of acute kidney injury and repair following acute kidney injury. They demonstrate that DA-PT cells accumulate after injury and persist following severe injury, potentially due to alteration of genes related to glutathione metabolism and ferroptosis. Their results are derived from single-cell RNAs sequencing and quantitative microscopic approaches in the mouse kidney. The studies from this work will have a significant impact not only to those studying acute kidney injury but also ischemic injury in other organ systems.

    Major strengths of this work include the comprehensive and complementary nature of the studies with pertinent in vivo models and data analysis of single cell RNA sequencing from kidney cells. The authors have achieved most of the aims of their study and the results mostly support their conclusions. The discussion is a nice summary of their work and compares their results to what is available in the literature.

    A major weakness of this work is the reliance on SOX9+ cells to represent DA-PT cells. This is relevant since their results show that less than 40% of the DA-PT cells express SOX9. SOX9 is not specific to DA-PT cells either, as they are seen in both PT cells and DCT1 cells as well. Additional clarification of these data would be important to enhacne the significance of this work.

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  4. Evaluation Summary:

    Ferroptotic cell death underlies tissue dysfunction inflicted by transient ischemia/reperfusion particularly in renal tissue. Here, the authors provide experimental evidence in animal models and human biopsies that mild and severe ischemic stress trigger distinctive cellular responses in proximal tubular cells which decide upon whether or not tissue may regenerate or fail. This is further corroborated in a genetic mouse model with mild ischemic stress-induced ablation of the key ferroptosis regulator glutathione peroxidase 4 (GPX4). These studies will be of significant interest both to those studying acute kidney injury and others interested in ischemic injury in other organ systems.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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