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

We appreciate Meng et al. for their thoughtful letter¹ on our JASN article.² We agree that large-scale, integrative multiomics analyses enable the discovery of previously unrecognized molecular connections across diverse biological layers, including proteins, genes, and metabolites. Motivated by this concept, our study combined single-cell transcriptomics with lipidomics to uncover a link between ferroptosis and dysregulated cholesterol metabolism. Through spatial transcriptomic mapping, we further identified the spatial localization of this process and delineated a pivotal role for the Cyp7b1/27-HC/ERα axis in mediating ferroptotic susceptibility within proximal straight tubular epithelial cells.²

As noted, ferroptosis was most prominent in injured proximal straight tubular epithelial cells characterized by downregulation of Cyp7b1 and upregulation of Acsl4 and Hmox1, consistent with heightened lipid peroxidation susceptibility. After performing within-cluster regressions on our published datasets (GSE267242),³ and adjusting for injury score, cell cycle phase, and mitochondrial read fraction, the inverse association between Cyp7b1 expression and the ferroptosis module in PST-4/PST-5 persisted (partial r=–0.45; 95% confidence interval, –0.41 to –0.34; regression β per 1-SD Cyp7b1=–0.42; 95% confidence interval, –0.46 to –0.32; P < 0.001). The exclusion of Hmox1 and Acsl4 from the ferroptosis module had a minimal effect (Δβ<0.02), and PST-5 maintained the highest ferroptosis score (decreasing only from 0.1783 to 0.1554), demonstrating that the association is robust and not solely dependent on these genes or global injury/dedifferentiation. These findings further support the notion that the Cyp7b1/27-HC/ERα axis independently modulates ferroptosis susceptibility in ischemic AKI.

In addition, we validated this association extensively at the protein level through multiple wet-lab experiments. While integrative multiomics analyses provide valuable hypotheses and reveal potential molecular correlations, discrepancies often exist between RNA and protein abundance, and statistical associations alone cannot establish causality. Therefore, functional validation remains essential to confirm the mechanistic relevance.

We also agree that distinguishing pathway specificity from injury-associated dedifferentiation is essential for interpreting cell death signatures.⁴ We are confident that our additional analysis reinforces the mechanistic inference that impaired cholesterol hydroxylation primes proximal straight tubular epithelial cells for ferroptotic injury, beyond general injury severity. We are grateful for the opportunity to clarify this point and for the constructive dialogue that advances understanding of lipid metabolism and regulated cell death in kidney injury.

Supplementary Material

jasn-37-649-s001.pdf ^{(1.4MB, pdf)}

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F503.

Author Contributions

Funding acquisition: Bi-Cheng Liu.

Investigation: Bi-Cheng Liu.

Writing – original draft: Yi-Lin Zhang.

Writing – review & editing: Bi-Cheng Liu, Bin Wang, Yi-Lin Zhang.

Funding

B.-C. Liu: National Key Research and Development Program (2022YFC2502500) and National Outstanding Doctor Program (CZXM-GSP-RC161).