Clarifying the Robustness of the Cyp7b1/27-Hydroxycholesterol Axis in Ferroptosis Susceptibility within Proximal Straight Tubules in Ischemic Acute Kidney Injury

In a recent JASN article, Zhang et al. elegantly integrated spatial single-cell transcriptomics with lipidomics to identify a Cyp7b1/27-hydroxycholesterol (27-HC) axis that primes proximal straight tubules for ferroptosis in ischemic AKI, corroborated by ANCA-associated vasculitis-Cyp7b1 rescue.1 This map of segment-specific injury biology is highly informative.2 We write with one focused, dataset-answerable question about the robustness of the ferroptosis signal in proximal straight tubules. In the article, ferroptosis is predominant in injured proximal straight tubules (PST) clusters, with high ACSL4 and HMOX1 and Cyp7b1 downregulation.1 Because ferroptosis gene modules often include HMOX1 and ACSL4—the very genes lying downstream or upstream of the proposed 27-HC/estrogen receptor alpha pathway—gene set composition could amplify the association.3 Within the public single-cell RNA sequencing (GSE267242) and spatial datasets accompanying the article, could the authors clarify whether the inverse association between Cyp7b1 expression and the ferroptosis module in PST-4/PST-5 and outer-stripe spots persists after (1) adjusting for the authors' published injury score (Supplemental Table 2), cell cycle phase, and mitochondrial read fraction and (2) recomputing the ferroptosis module excluding HMOX1 and ACSL4? A within-cluster linear/partial-correlation or regression, with effect sizes and confidence intervals, would address this.4 If the association remains materially unchanged, it would strengthen the inference that the Cyp7b1/27-HC–estrogen receptor alpha axis confers ferroptosis susceptibility beyond global injury/dedifferentiation.5 If it attenuates, readers would better understand how much of the signal reflects injury severity rather than pathway specificity. Either way, this sensitivity analysis is feasible with the published datasets and would sharpen the mechanistic message for the community. We congratulate the authors on this important contribution and look forward to their clarification.