Dietary Choline Deprivation Exacerbates Kidney Injury in Streptozotocin-Induced Diabetes in Adult Rats^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study explores how choline deprivation exacerbates kidney injury in a diabetic model, which is highly relevant to understanding diabetic nephropathy. I have several suggestions to improve this manuscript.

Minor:

1. Table 1 is repeated.

2. The renal necrosis and fibrosis sites identified in histological staining should be clearly marked or annotated to enhance interpretability.

Major:

1. The immunohistochemistry images, especially for KIM-1 and VEGF-A, are of poor quality and lack sufficient clarity. Consider supplementing these with quantitative data from qPCR or Western Blot analysis for a more robust validation of protein expression levels.

2. A rescue experiment that includes a non-diabetic, choline-supplemented group could provide better insight into the protective effects of choline supplementation.

3. While the study mentions mechanisms like oxidative stress and fibrosis pathways, deeper exploration into these pathways (e.g., specific signaling cascades) using additional assays could strengthen the manuscript.

Author Response

REVIEWER #1

Comment 1: ‘’Comments and Suggestions for Authors.

The study explores how choline deprivation exacerbates kidney injury in a diabetic model, which is highly relevant to understanding diabetic nephropathy. I have several suggestions to improve this manuscript.’’

Response 1: We thank the reviewer for the very helpful and constructive comments which helped us to improve the manuscript.

 

Comment 2: Minor: Table 1 is repeated.

Response 2: Apologies, we have corrected our mistake.

 

Comment 3: Minor: The renal necrosis and fibrosis sites identified in histological staining should be clearly marked or annotated to enhance interpretability.

Response 3: As requested, we have addressed this in the revised figures.  

 

Comment 4: Major:The immunohistochemistry images, especially for KIM-1 and VEGF-A, are of poor quality and lack sufficient clarity. Consider supplementing these with quantitative data from qPCR or Western Blot analysis for a more robust validation of protein expression levels.

 Response 4: We acknowledge that the quality of the images used in this study was suboptimal. Additionally, KIM-1 sections were prepared from frozen samples, which made it more challenging to obtain high-quality images. We request the indulgence of the reviewer because we can only address this issue in future studies with incorporating quantitative validation methods, such as qPCR and Western Blot analysis. We agree this will provide more robust and reliable data on protein expression levels. We have highlighted this in the limitation section in the revised manuscript.

 

Comment 5: Major: A rescue experiment that includes a non-diabetic, choline-supplemented group could provide better insight into the protective effects of choline supplementation.

Response 5: We thank the Reviewer for the insightful point. While we recognise the substantial value of such a comparison, our study was specifically focused on exploring the impact of choline deficiency on kidney pathology in the context of diabetes. The combined common conditions remains unexplored. We are unfortunately not in a position at the moment to conduct additional experiments but we have addressed this point in the limitation section.

 

Comment 6: ‘’3. While the study mentions mechanisms like oxidative stress and fibrosis pathways, deeper exploration into these pathways (e.g., specific signaling cascades) using additional assays could strengthen the manuscript.’’

Response 6: Thank you for your valuable comment, we have incorporated relevant information into the text. Unfortunately, we are unable to perform additional assays at this stage; however, a more in-depth exploration of these pathways is one of our next objectives.

Reviewer 2 Report

Comments and Suggestions for Authors

Revision of the manuscript entitled:

 

“Dietary Choline Deprivation Exacerbates Kidney Injury in 2 Streptozotocin-Induced Diabetes in Adult Rats” 

    

Authors: Ahmed W. Al-Humadi, Carel W. le Roux, Neil Docherty, Werd Al-Najim, Martin Tze Wah Kueh, Andreas Lazaris, and Charis Liapi

 

 

I am very happy to have the opportunity to revise such an interesting paper which gives very interesting information about synergistic effect of Choline deprivation and diabetes mellitus type 2 on kidney biochemistry, histopathology, and immunohistochemistry. Although findings would be of great benefit in the field of the treatment of chronic diabetic kidney disease, the manuscript needs to be improved considerably, especially in the material and methods section.

 

Here are my major observations:

1. Minor English Editing: Some sentences need slight refinement for clarity and correctness.

2. Technical Improvements:

    Line 34: Ensure consistent text color.

    Line 35: Should this refer to "mesangial expansion"?

    Line 129: Brackets are missing; please address this.

3. Study Duration: Could you explain why the study was conducted specifically for 5 weeks?

4. Histopathological Classification (Line 131): Provide a more detailed description of how you assessed the histopathological classification.

5. Absolute Injury Score (Line 131): Elaborate on how the absolute score for histopathological injury classification was determined.

6. Methods Section: This section should be rewritten to be more concise and precise. Include all necessary details to ensure clarity and reproducibility of the measurements.

7. Results Explanation: The results section is thorough and well-detailed. However, the Materials and Methods section should explain the assessment methods for the tubular necrosis score, mesangial matrix expansion score, and renal fibrosis score.

 

8. Figures (HE, PAS, Masson Trichrome, IHC): Improve the quality by using higher magnification and reducing background noise to highlight morphological changes. Add scale bars to all figures.

Comments for author File: Comments.pdf

Author Response

REVIEWER # 2:

Comment 1: ‘’I am very happy to revise such an interesting paper which gives very interesting information about synergistic effect of Choline deprivation and diabetes mellitus type 2 on kidney biochemistry, histopathology, and immunohistochemistry. Although findings would be of great benefit in the field of the treatment of chronic diabetic kidney disease, the manuscript needs to be improved considerably, especially in the material and methods section.’’

Response 1: We sincerely thank Reviewer 2 for the kind and constructive comments.

 

Comment 2: ‘’Minor English Editing: Some sentences need slight refinement for clarity and correctness.’’

Response 2: The entire manuscript has been carefully revised to enhance clarity, readability, grammar, and sentence structure. We believe these changes have strengthened the overall quality of the manuscript.

 

Comment 3: ‘’2. Technical Improvements: Line 34: Ensure consistent text color.’’

Response 3: As requested, we have made the correction.

 

Comment 4: ‘’Line 35: Should this refer to "mesangial expansion"?’’

Response 4: Thank you, the reviewer is correct and have addressed this in the revised manuscript. 

 

Comment 5: ‘’Line 129: Brackets are missing; please address this.’’

Response 5: Done.

 

Comment 6: ‘’Study Duration: Could you explain why the study was conducted specifically for 5 weeks?’’

Response 6: The rationale for the 5-week duration of this study is based on our previous research, as well as that of others, investigating the impact of choline deficiency on the liver, heart, and brain in the absence of comorbidities. However, this study aims to explore the impact of choline deficiency on kidney pathology in the context of diabetes, a topic that, to our knowledge, remains largely unexplored. In this work, we investigate whether the findings from our earlier studies (Al-Humadi et al., 2021; Al-Humadi et al., 2018; Strilakou et al., 2013; Strilakou et al., 2016), which demonstrated the effects of choline deficiency on cardiac function and structure, are also applicable to renal injury under diabetic conditions. The 5-week duration was initially chosen as an appropriate timeframe to detect meaningful changes in kidney morphology and function while simulating a chronic state of choline deprivation in the context of diabetes. As noted on the discussion section (limitation), we acknowledge that further studies, involving extended duration, are necessary to fully elucidate these effects."

 

Comment 7: Histopathological Classification (Line 131): Provide a more detailed description of how you assessed the histopathological classification.

Response 7: We thank Reviewer 2 for the valuable point, histopathological classifications were clarified in our revised manuscript.

 

Comment 8:  ‘’Absolute Injury Score (Line 131): Elaborate on how the absolute score for histopathological injury classification was determined.’’

Response 8: Thank you, information regarding the renal injury scores has been incorporated into the revised manuscript.

 

Comment 9: ‘’Methods Section: This section should be rewritten to be more concise and precise. Include all necessary details to ensure clarity and reproducibility of the measurements.’’

Response 9: As requested, the methods section has been revised to be more concise and precise, taking the reviewer’s comments into account.

 

Comment 10: ‘’Results Explanation: The results section is thorough and well-detailed. However, the Materials and Methods section should explain the assessment methods for the tubular necrosis score, mesangial matrix expansion score, and renal fibrosis score.’’

Response 10: Thank you for the helpful comment. The Materials and Methods section has been edited, the relevant information has been included.

 

Comment 11: ‘’Figures (HE, PAS, Masson Trichrome, IHC): Improve the quality by using higher magnification and reducing background noise to highlight morphological changes. Add scale bars to all figures.’’

Response 11: We have added scale bars. However, we would like to apologise for the suboptimal quality of some of the images presented in our manuscript. Unfortunately, due to technical limitations during the image acquisition process, the quality of certain sections was not as high as we would have preferred. We understand the importance of clear, high-quality images in accurately conveying our findings, and we regret that these images may have detracted from the overall presentation of our results. We appreciate your consideration of this limitation in our study.

We are committed to take extra care in optimising imaging techniques to ensure better resolution and clarity in future studies. We appreciate the Reviewer’s understanding and constructive feedback.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no more comments.