PONE-D-19-26349

Change of surfactant protein D and A after renal ischemia reperfusion injury

PLOS ONE

Dear Dr Gil,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by two experts and  they send mixed responses. All of their comments must be addressed during revision and those are provided below.

We would appreciate receiving your revised manuscript by Dec 15 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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3.  As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist, a document that  aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. Please also include the approval number given to your study by your ethics committee.

4. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.3892/or.2018.6381

https://doi.org/10.1371/journal.pone.0178665

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled “Change of surfactant protein D and A after renal ischemia reperfusion injury” suggested a link between serum surfactant protein and renal ischemia reperfusion models. Furthermore, increased lung inflammation and apoptosis were observed due to change of lung tight-junction protein. The authors demonstrated the cross-talk between organs (kidney and lung), however, several concerns must be addressed.

<major concerns="">

- The information in the manuscript does not provide a very well-explainable data between AKI and surfactant protein. Albeit further study will be required in the future, certain conclusion must be given in the manuscript. Therefore, it is pretty tricky to consider the overall ideas the authors provided. The authors need to provide schematic overview of these results for the readers to understand easily.

- There are too much information on materials and methods. The authors need to make it simplify.

- General figure image quality is too poor. It is very difficult to recognize clearly (histology, graph and western blot). It must be addressed to increase image quality (more than 300DPI).

- In results part, the “sub-title” must summarize the results obtained. There is no information of the sub-title the authors provided in the manuscript. It will be re-organized.

- The methods used are described in FIGURE LEGEND. Do not state the results in the FIGURE LEGEND.

For example,

(FIG 1A) Renal function in C57BL/6 mice renal ischemia reperfusion blood urea nitrogen (BUN) and creatinine “increased” --> “evaluated”;

(FIG 1B) Tubular injury necrosis scores for PAS-stained kidney sections showed increased tubular injury in ischemic AKI --> kidney sections were subjected to PAS-staining and histological changes were scored.

- Fig3B and Fig4 Western blot images, the bands were cut group by group. The authors must provide the entire row immune-blotting bands. Do not cut (or split) the Western bands.

<minor concerns="">

- Figure1A, the order of graphs (BUN and creatine) should be changed. The manuscript stated serum creatinine and BUN (in the results). Try to make the consistency.

- Figure1B, please indicate the areas of tubular damage (i.e. using arrow head)

- Line 182, “protein in BAL increased in the AKI”. Which protein? How to measure? Or any protein changes between shame and AKI models?

- There is no scale bars on Figures1,3 and5 (microscopic images)

- In figure2, put Blood serum on top of the graphs to recognize them easily.

- In figure3A, how to measure neutrophil infiltration? By FACS or by count? If it was evaluated by count, only 1-2 neutrophils in the field? Make them more clarify.

-In figure4 Western blotting, there is no information about the bands. Once again, do not split the western blot images.

Reviewer #2: The authors reported changes of SP-A and SP-D in an AKI mice model at different time points and compared serum and kidney SP-A & SP-D changes to other organ specific markers. They concluded that kidney-originated SP-A/D changes could not be ruled out and thus the two markers are unsuitable for determining AKI-related lung injury. The overall experimental design is adequate, however more details in animal experiments are welcomed. I have a few comments/questions:

In Materials and methods, please indicate sex of animals used.

How was animal sacrifice performed? Spine dislocation or anesthetic overdose? Please specify.

Please define ‘80% return’ in BAL collection paragraph and specify pH or product number of PBS used.

Did you conduct PBS flush of lung vasculature before extracting BAL? why or why not?

Which parts of centrifugated serum or BAL samples were kept? Please specify.

In results:

In SP-A and SP-D assays, error bars are relatively outstanding. Specifically, Lung SP-A protein assay displayed inconsistency in 24h sham group, can the author address the cause of this anomaly? 4hr sham groups and 24h sham group underwent similar surgical procedures, and yet SP-A protein in kidney shows reduction in 24hr sham vs. 4hr sham. Which leads to suspicion that the ‘sham’ surgery is too invasive and disruptive to kidney function, Can the authors please address this phenomenon?

In some data presentation, one group presents much more substantial error bar than other groups. In common knowledge, age and body weight controlled mice are expected to have very consistent biological and biochemical profile under consistent modeling technique. I have to say that the data quality is questionable.

In multiple graphs, SP-A and SP-D shows different trends, it would appear to me that SP-D responds in latent phase while SP-A has more timely response and increased very soon after ischemia reperfusion injury occurs. Why we should look at both targets at the same time, and how do we interpret the difference in their responses? I hope the authors can give a bit discussion in this aspect.

The topic brought up by the authors warrants attention, however the results are of less that satisfying quality. It would be great if well controlled experiments are performed and consistent data is presented, then the same conclusions will be much more convincing.

 </minor></major>

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-26349R1

Change of surfactant protein D and A after renal ischemia reperfusion injury

PLOS ONE

Dear Dr Gil,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by same reviewers and we received positive feedback from them. However, numerous errors and minor technical questions raised by one of the reviewers.

We would appreciate receiving your revised manuscript by Jan 20 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. In Fig1A, Y axis of the graphs, mg/dL and mg/dl are mixed. Make them consistent.

2. In the manuscript, put the full name of ATN (might be “Acute tubular necrosis”), related with Fig1B

3. In Fig3A, H&E staining for neutrophil is not clear to determine NP infiltration. The evaluation of NP infiltration was done by pathologist according to the authors explanation and it was fully understandable. However, please perform MPO staining to confirm NP infiltration on those sites and put that data on Supporting figure?

4. In Fig4 (western blot), JAMA-1 or JAM-A? clarify

5. In Fig4, Claudin 3 , Claudin 4, Claudin 18 and Claudin-3, Claudin-4, Claudin-18 are mixed. Make them consistent.

6. In Fig6, the staining SP-A and SP-D was not still clear. Please count SP-A and SP-D positive cells and add one other graph how many positive cells were counted in each group.

Reviewer #2: The authors has included additional materials supplementing the findings and completing the data presentations. previous concerns are addressed by additional descriptions or discussion.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Change of surfactant protein D and A after renal ischemia reperfusion injury

PONE-D-19-26349R2

Dear Dr. Gil,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Partha Mukhopadhyay, Ph.D.

Section Editor

PLOS ONE

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