Vitamin B12 Protects the Exacerbated Ischemia–Reperfusion Injury-Induced Chronic Kidney Disease in Mice with Genetically Increased Elmo1

Round 1

Reviewer 1 Report

The study addresses an important topic — the mechanisms driving AKI-to-CKD progression and the potential protective role of vitamin B12. However, while the experimental design is ambitious and the findings promising, the manuscript has several weaknesses that limit its impact and translational relevance.

- The entire study is based on C57BL/6J mice with genetically increased Elmo1. Although this model is relevant, the lack of validation in additional strains or in vitro systems weakens the generalizability.

- Given the complexity of human CKD, conclusions about vitamin B12 as a therapeutic option appear premature.

- The administered dose of vitamin B12 (50 mg/L in drinking water) is exceptionally high compared to physiological or clinical supplementation.

- The authors acknowledge possible toxic metabolite accumulation in patients with impaired renal clearance, but no systematic analysis of potential side effects is presented. This raises concerns about clinical applicability.

- While the study identifies oxidative stress as a central mechanism, the molecular exploration remains limited to canonical antioxidant/pro-oxidant genes (Sod1, Gpx1, Nox2).

- Broader redox and signaling pathways (e.g., Nrf2, mitochondrial metabolism, DNA damage response) are not examined, leaving the mechanistic explanation incomplete.

- The observation that female mice display milder CKD-like phenotypes is intriguing. However, the authors treat this as a side note rather than a core finding.

- No mechanistic analysis is offered, missing an opportunity to address a highly relevant dimension of CKD research.

- Certain results (e.g., plasma endothelin-1 levels, inflammatory gene profiles) are inconsistent or lack statistical robustness, yet they are not critically discussed.

- The discussion often overstates conclusions, particularly regarding translational readiness of vitamin B12 therapy.

Author Response

- The entire study is based on C57BL/6J mice with genetically increased Elmo1. Although this model is relevant, the lack of validation in additional strains or in vitro systems weakens the generalizability.

Response:

Thank you for pointing this out. We agree that relying on a single mouse strain limits the generalizability of our findings. We selected the C57BL/6J (B6) strain due to its well-characterized genetic background and wide use in disease modeling, which facilitates reproducibility and interpretation. In the current work on the role of increased Elmo1 gene expression levels in kidney CKD progression, we decided to focus on animals with overexpression because we already have established that Elmo1 low expressions are protected in the diabetic kidney disease. To address this concern, we have initiated plans to expand our validation to additional genetic backgrounds in future work.

Importantly, we have also performed complementary in vitro experiments using mouse proximal tubular cells transfected with an Elmo1-overexpression plasmid. When exposed to low-nutrient media and hypoxic conditions with hypoxia chamber, these cells exhibited a pattern consistent with our in vivo findings, showing exacerbated oxidative stress and impaired mitochondrial and redox homeostasis in the Elmo1^H/H background. These parallel results strengthen the biological relevance of our observations beyond the single-strain model.

 

- Given the complexity of human CKD, conclusions about vitamin B12 as a therapeutic option appear premature.

Response:

Thank you for this valuable comment. We agree that it is premature to make definitive claims about vitamin B12 as a therapeutic intervention in human CKD. Accordingly, we have revised the wording in lines 467–472 to more cautiously describe the role of B12 as potentially protective rather than therapeutic, and we now emphasize the need for further validation in translational and clinical studies.

 

- The administered dose of vitamin B12 (50 mg/L in drinking water) is exceptionally high compared to physiological or clinical supplementation.

- The authors acknowledge possible toxic metabolite accumulation in patients with impaired renal clearance, but no systematic analysis of potential side effects is presented. This raises concerns about clinical applicability.

- The discussion often overstates conclusions, particularly regarding the translational readiness of vitamin B12 therapy.

 

Response:

We appreciate this important observation. We are aware that the administered dose of vitamin B12 is higher than physiological supplementation levels. However, in our previous studies using similar or higher doses in mouse models of kidney and metabolic complications, no adverse effects were observed. This is consistent with vitamin B12’s water-soluble nature, which allows excess amounts to be efficiently excreted.

We have examined the dose in mice earlier studies and the current dose do not induce any adverse effects. We reduced dose further, yet it is a physiologically high dose, and it is important to adjust carefully check in humans. To address the reviewer’s concern, we have expanded the discussion (lines 447-449, 454-459) to highlight that although no toxicity was detected in our prior or current experiments, the optimal dosing and treatment duration should be systematically evaluated in future studies, particularly in the context of impaired renal filtration and clearance.

 

While the study identifies oxidative stress as a central mechanism, the molecular exploration remains limited to canonical antioxidant/pro-oxidant genes (Sod1, Gpx1, Nox2).

- Broader redox and signaling pathways (e.g., Nrf2, mitochondrial metabolism, DNA damage response) are not examined, leaving the mechanistic explanation incomplete.

Response:

We appreciate the reviewer’s constructive suggestion. In response, we have expanded our analysis to include Nrf2 (gene name Nfe2l2), a key transcriptional regulator of redox balance. The new qRT-PCR results (lines 245–250; Figure 3G) show that Elmo1 overexpression did not significantly alter Nrf2 expression, suggesting that the observed oxidative imbalance may occur downstream or independently of this canonical pathway. Fig 6D. demonstrate that vitamin B12 treatment enhances Nrf2 expression and partially restores antioxidant capacity. We also noted that Nfe2l2 appeared in the ischemic kidney heatmap but displayed a mixed expression pattern, further supporting the complexity of its regulation under oxidative stress.

In addition, we incorporated two new heatmaps of DNA damage response and mitochondrial function–related genes (lines 263–273; Supplementary Figures 6A-6C). These results demonstrate that Elmo1^H/H kidneys exhibit enhanced DNA damage signaling and impaired mitochondrial metabolism—both consistent with oxidative stress–induced dysfunction. We have also noted in the Discussion that further mechanistic studies are warranted to clarify the regulatory links between Elmo1, Nrf2 activity, and redox-driven metabolic reprogramming during AKI-to-CKD progression.

 

- The observation that female mice display milder CKD-like phenotypes is intriguing. However, the authors treat this as a side note rather than a core finding.

Response:

We appreciate the reviewer’s insightful observation. We agree that the sex-related differences in disease manifestation are an important aspect of our findings. Accordingly, we have revised the manuscript to emphasize this point more clearly and added a new paragraph in the Discussion (lines 392–401). The added section highlights the potential role of sexual dimorphism in the progression of kidney injury and discusses distinct pathogenic mechanisms that may underlie the milder CKD-like phenotype observed in female mice. We also note that further investigation into sex-specific molecular pathways could provide valuable insights into differential disease susceptibility and therapeutic response.

- No mechanistic analysis is offered, missing an opportunity to address a highly relevant dimension of CKD research.

Response: We thank the reviewer for this valuable comment. We agree that elucidating the underlying mechanisms of Elmo1 overexpression in CKD progression is critical. To address this, we have expanded the Discussion (lines 406–412) to include a detailed section outlining potential molecular pathways through which Elmo1-Rac1-NADPH Oxidase may modulate oxidative stress, mitochondrial dysfunction, and inflammatory signaling. Additionally, we have added a paragraph (lines 419-423) discussing future research directions, including targeted transcriptomic and proteomic analyses to define downstream mediators and signaling cascades involved in Elmo1-driven kidney injury.

- Certain results (e.g., plasma endothelin-1 levels, inflammatory gene profiles) are inconsistent or lack statistical robustness, yet they are not critically discussed.

We appreciate the reviewer’s thoughtful comment. We acknowledge that the inflammatory and fibrosis gene profiles were discussed mainly as supporting observations to the oxidative stress findings, which represent the central focus of this study. Our primary goal was to elucidate the chronic oxidative stress and maladaptive repair mechanisms driven by Elmo1 overexpression and the long-term restorative effects of vitamin B12 treatment—a novel aspect that, to our knowledge, has not been previously reported.

Regarding endothelin-1 (ET-1), we included this analysis because of its established role in vascular tone regulation and endothelial dysfunction, which may exacerbate kidney injury. However, given the limited statistical robustness of these findings, we have now clarified this point in the revised text and moved related data to the Supplementary Information for transparency and completeness.

 

Reviewer 2 Report

I am honored to have reviewed this well-designed, well-powered study with potentially important clinical implications. This article clearly elucidates the process of ELMO1 exacerbating the transformation of renal ischemia-reperfusion injury (IRI) to chronic kidney disease (CKD) through oxidative stress pathway, and innovatively validates the therapeutic potential of high-dose vitamin B12 as an effective antioxidant. The scientific logic of the research is clear, and the chain of in vivo experimental evidence is complete, ranging from phenotype (renal function, tissue morphology) to mechanism (oxidative stress, fibrosis, inflammation). However, from the perspective of journal publication standards, there are still many problems in the manuscript that need to be improved, such as incomplete presentation of some experimental results, insufficient depth of mechanism discussion, lack of detail description, non-standard literature citation and format, which need to be further modified and optimized to meet the requirements of high-level journal publication.

1. Existing studies have only preliminary relationship between Elmo1 and oxidative stress and the antioxidant effect of vitamin B12. It is necessary to further explore the specific molecular mechanism of Elmo1 regulating ROS generation through Rac1-NADPH oxidase pathway (such as detecting Rac1 activity and NADPH oxidase isoforms expression). At the same time, whether vitamin B12 supplementation exerts its renoprotective effect through other pathways (such as the regulation of one-carbon metabolism) besides scavenging superoxide anions (such as the detection of the activity or expression of key enzymes of one-carbon metabolism).
2. All citations were checked. Ensure that conform to the requirements of the journal of the Antioxidants (such as the literature [3] the doi format is "https://doi.org/10.14814/phy2.15211" with the other documents to form a "10.14814 / phy2.15211"), Supplement the literature cited in this article but missing in the reference list (such as "as previously described [14]", it is necessary to confirm whether [14] accurately corresponds to relevant experimental methods), and cite the latest research results in this field in the past 2 years (2023-2025) to enhance the timeliness and relevance of the research. Such as PMID: 34237625, PMID:38064284, PMID: 39477605.
3. Lines 41-42 "CKD affects approximately 14% of adults in the United States and is more prevalent among older adults, women, and non-Hispanic black individuals [1] ", after citing literature [1], it is suggested to supplement the time context of the data (e.g., literature [1] published in 2022, The data are from the 2022 Chronic Kidney Disease Epidemiology Report in the United States.

1. In experimental methods, it is recommended to supplement the source of mice to increase the traceability of the source of experimental animals.
2. Lines 97-98 "Semi-quantification of tubular injury was performed by an investigator who was blinded to the experimental It is necessary to clarify the semi-quantitative scoring standard of renal tubular injury (such as scoring from renal tubular dilatation, vacuolar degeneration, brush border loss, etc., with a full score of X by referring to [specific scoring methods literature]) to avoid subjective errors affecting the credibility of the results.
3. There are a few grammatical errors, unclear statements or inconsistencies in the article, which need to be polished.
4. The data in Figure 7 show a lighter female phenotype, which is an interesting finding. However, it is currently only used as a secondary outcome. It is recommended that more space be devoted to the underlying mechanisms in the discussion.
5. the "completely reversed the severity of CKD in Elmo1H/H mice." in line 356 is too absolute. It is suggested that the author revise the description again.
6. It is suggested that a paragraph be added at the end of the discussion to briefly explain the limitations of this study.

Author Response

1. In experimental methods, it is recommended to supplement the source of mice to increase the traceability of the source of experimental animals.

Response: Thank you for this helpful suggestion. We have now added the source and genetic background of the mice in the Methods section (lines 72–75). In the method section, we expanded and talked about the details of the generation and characterization of this Elmo1-overexpressing strain, thereby improving traceability and methodological transparency.

 

2. Lines 97-98 "Semi-quantification of tubular injury was performed by an investigator who was blinded to the experimental It is necessary to clarify the semi-quantitative scoring standard of renal tubular injury (such as scoring from renal tubular dilatation, vacuolar degeneration, brush border loss, etc., with a full score of X by referring to [specific scoring methods literature]) to avoid subjective errors affecting the credibility of the results.

Response: Thank you for this helpful suggestion. We have now clarified the semi-quantitative scoring criteria in the Methods section (lines 101–104). The scoring system was adapted from the Banff Working Classification for Kidney Transplant Pathology, following the interstitial fibrosis and tubular atrophy (IFTA) grading scheme. The updated description includes the specific histopathologic features evaluated (tubular dilatation, vacuolar degeneration, and brush border loss) and the corresponding reference citation to ensure methodological transparency and reproducibility.

3. There are a few grammatical errors, unclear statements or inconsistencies in the article, which need to be polished.

We appreciate the reviewer’s careful reading and helpful feedback. We have thoroughly revised the manuscript for grammar, clarity, and consistency throughout. Several sentences were rewritten to improve readability, and minor typographical errors were corrected. The revised version has been carefully proofread by all authors and reviewed by a native English speaker to ensure clarity and linguistic accuracy.

4. The data in Figure 7 show a lighter female phenotype, which is an interesting finding. However, it is currently only used as a secondary outcome. It is recommended that more space be devoted to the underlying mechanisms in the discussion.

 

Response: We thank the reviewer for highlighting this important point. We agree that the milder CKD-like phenotype observed in female mice is an intriguing finding deserving further emphasis. Accordingly, we have added a new paragraph in the Discussion (lines 392–402) to elaborate on the potential mechanisms of sexual dimorphism in Elmo1-associated kidney injury, including differences in hormonal regulation and disease progression This addition expands on Figure 7 and provides a broader context for understanding sex-specific susceptibility and protective mechanisms in kidney disease progression.

 

5. the "completely reversed the severity of CKD in Elmo1H/H mice." in line 356 is too absolute. It is suggested that the author revise the description again.

Response: We appreciate the reviewer’s thoughtful suggestion. We have revised the statement to avoid overinterpretation. The updated text (lines 381-382) now reads:

“Supplementation immediately after surgery showed protection against the progression from AKI to CKD in Elmo1^H/H mice.”

 

6. It is suggested that a paragraph be added at the end of the discussion to briefly explain the limitations of this study.

Response: We appreciate the reviewer’s helpful suggestion. A new paragraph summarizing the key limitations of the study has been added to the Discussion section (lines 460-472). This section now acknowledges the constraints of using a single mouse strain, the limited scope of mechanistic exploration, and the high experimental dose of vitamin B12, while emphasizing the need for further multi-strain, long-term, and translational studies to validate the findings.

 

I am honored to have reviewed this well-designed, well-powered study with potentially important clinical implications. This article clearly elucidates the process of ELMO1 exacerbating the transformation of renal ischemia-reperfusion injury (IRI) to chronic kidney disease (CKD) through oxidative stress pathway, and innovatively validates the therapeutic potential of high-dose vitamin B12 as an effective antioxidant. The scientific logic of the research is clear, and the chain of in vivo experimental evidence is complete, ranging from phenotype (renal function, tissue morphology) to mechanism (oxidative stress, fibrosis, inflammation). However, from the perspective of journal publication standards, there are still many problems in the manuscript that need to be improved, such as incomplete presentation of some experimental results, insufficient depth of mechanism discussion, lack of detail description, non-standard literature citation and format, which need to be further modified and optimized to meet the requirements of high-level journal publication.

1. Existing studies have only preliminary relationship between Elmo1 and oxidative stress and the antioxidant effect of vitamin B12. It is necessary to further explore the specific molecular mechanism of Elmo1 regulating ROS generation through Rac1-NADPH oxidase pathway (such as detecting Rac1 activity and NADPH oxidase isoforms expression). At the same time, whether vitamin B12 supplementation exerts its renoprotective effect through other pathways (such as the regulation of one-carbon metabolism) besides scavenging superoxide anions (such as the detection of the activity or expression of key enzymes of one-carbon metabolism).

Response: We thank the reviewer for these valuable suggestions. We have expanded the Discussion (lines 405–412) to include a more detailed consideration of the Rac1–NADPH oxidase pathway as a potential mechanism by which Elmo1 may regulate ROS generation from the published data from our lab’s previous work. We also acknowledge that this is a complex system potentially affecting multiple organs and systemic responses beyond local renal injury.

Regarding one-carbon metabolism, we had previously discussed its importance in the Discussion and have further emphasized its potential role in mediating the broader metabolic effects of vitamin B12 (lines 434–439). Our laboratory’s prior work in diabetic cardiomyopathy models has explored several one-carbon pathway genes in livers, and we plan to extend this investigation to the kidney context in future studies, as vitamin B12’s function likely extends well beyond its antioxidant properties.

 

2. All citations were checked. Ensure that conform to the requirements of the journal of the Antioxidants (such as the literature [3] the doi format is "https://doi.org/10.14814/phy2.15211" with the other documents to form a "10.14814 / phy2.15211"), Supplement the literature cited in this article but missing in the reference list (such as "as previously described [14]", it is necessary to confirm whether [14] accurately corresponds to relevant experimental methods), and cite the latest research results in this field in the past 2 years (2023-2025) to enhance the timeliness and relevance of the research. Such as PMID: 34237625, PMID:38064284, PMID: 39477605.

Response: We thank the reviewer for these detailed and helpful comments. We have reviewed and standardized all reference formats to conform to the journal’s citation style, including correction of the DOI formatting for references [3], [13], [34], and [47]. Reference [50] does not include a DOI due to its age and archival source.

We also confirmed that reference [14] accurately corresponds to the ischemia–reperfusion injury (IRI) model used in this study, ensuring methodological consistency with our previous experimental design, which allows direct comparison between acute and chronic injury outcomes.

To improve the timeliness and relevance of the manuscript, we have incorporated recent studies (2023–2025), including a newly added reference [48] discussing ROS signaling and cell death mechanisms relevant to our findings. Some older references were retained intentionally to preserve continuity in the discussion of disease progression and established models.

 

3. Lines 41-42 "CKD affects approximately 14% of adults in the United States and is more prevalent among older adults, women, and non-Hispanic black individuals [1] ", after citing literature [1], it is suggested to supplement the time context of the data (e.g., literature [1] published in 2022, The data are from the 2022 Chronic Kidney Disease Epidemiology Report in the United States.

Thank you for this helpful suggestion. We have revised the sentence to include the time context and data source. The updated text now reads:

“CKD affects approximately 14% of adults in the United States and is more prevalent among older adults, women, and non-Hispanic Black individuals, according to the 2022 Chronic Kidney Disease Epidemiology Report [1].”

This revision clarifies both the publication year and the data source for improved transparency.

Round 2

Reviewer 1 Report

I would like to thank the authors for addressing the raised comments. The manuscript has improved.

.

Reviewer 2 Report

I have no further questions.

I have no further questions.