PONE-D-22-09686Angiotensin-(3–4) normalizes the elevated arterial blood pressure and abnormal Na+/energy handling associated with chronic undernutrition by counteracting the effects mediated by type 1 angiotensin II receptorsPLOS ONE

Dear Dr. Vieyra

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Xianwu Cheng, M.D., Ph.D., FAHA

Academic Editor

PLOS ONE

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Additional Editor Comments:

The both reviewers have concerned statistical analysis. Thus, the authors should address all of these comments satisfactory before resubmission.

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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: Yes

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

Reviewer #1: No

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: No

Reviewer #2: Yes

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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: General Comments

In this study, the authors investigated the mechanisms of bodily Na+ handling and high systolic blood pressure in the rat model feeding a multideficient diet. They used two renin-angiotensin-aldosterone system (RAAS)-related drugs, losartan as an antagonist of AT1R, and Ang-(3–4) as an “allosteric enhancer” of AT2R .

They concluded that the chronic administration of a multideficient diet with a low content of protein and very poor quality is the primary cause – rather than excess Na+ – in the pathogenesis of hypertension in this rat model, by simultaneously targeting the Ang II�AT1R axis of local RAAS in kidney, the central nervous system (especially centers of food satiety, Na+ hungry control and cardiovascular regulation), and possibly bodily Na+ distribution and structural modifications of the cardiovascular system itself.

Specific comments.

1) P values are presented as four decimal places, which is meaningless. This is animal study, therefore P<0.01, P<0.001 or two decimal places is enough. See the article in PloS One reported from your laboratory (PLoS One. 2014 Jul 1;9(7):e100410).

2) Presentation of Figures are not appropriate. In Fig 1, for example, the same values of CTR (-/-)and RBD (-/-) columns show 4 columns in the same figure.

3) Also, the authors misunderstand ANOVA. ANOVA is applied to estimate the strictly correct variance of the parameter of interest to avoid screwed variances. Therefore, all groups in Fig1 must be put in the same ANOVA for calculation. The authors cannot use Student t-test for analysis to compare two groups of several groups.

4) Since there are no restrictions on the number of figures, it is better not to use supplementary figures.

5) Did the authors check the yield or purity of plasma membrane-rich fraction prepared for each ATPase activity from kidney proximal tubule cells among groups.

6) Manuscript text should be double-spaced according to instruction for authors.

Reviewer #2: In the current study, the authors investigated the mechanism of hypertension associated with chronic undernutrition. This study provides evidence that the chronic administration of a multi-deficient diet with a low content of protein of very poor quality is the primary cause – rather than excess Na+ in the pathogenesis of hypertension in undernourished rats, by simultaneously targeting the Ang II-AT1R axis of local RAAS in kidney, the central nervous system, and possibly bodily Na+ distribution and structural modifications of the cardiovascular system itself. I believe this study sheds light on the role of Ang II-AT1R axis and Ang II-AT2R axis in hypertension under the chronic undernutrition. However, I have some questions that needed to be answered.

1. Do the changes caused by RBD in the Wistar rats resemble that in human being, including Body weight, energy intake, Na+ ingestion，Na+ excretion, hypertension extent ?

2. Figure 7，the elevated SBP is almost normalized by Losartan in RBD group. Ang-(3-4）also has an hypotensive effect in RBD. Why the SBP seemed to increase SBP in the combination therapy group (Lorsartan+Ang-(3-4）) as compared to the single therapy group (Lorsartan or Ang-(3-4）) under RBD？

3. In figure 11-12，the ouabain-sensitive Na+ ATPase and the ouabain-resistant Na+ ATPase activities changes differently under RBD. What’s the different roles between the two kinds of Na+ ATPase? Please give more explanation in the Discussion.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-09686R1Angiotensin-(3–4) normalizes the elevated arterial blood pressure and abnormal Na+/energy handling associated with chronic undernutrition by counteracting the effects mediated by type 1 angiotensin II receptorsPLOS ONE

Dear Dr. Vieyra 

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.

Please submit your revised manuscript by Sep 04 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Xianwu Cheng, M.D., Ph.D., FAHA

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

None.

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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: All comments have been addressed

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

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

Reviewer #1: I Don't Know

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

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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

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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: The revised version has been significantly improved.

RE: P values in the Figures.

"P> 0.05" is not appropriate because the authors declare that "significant difference was set to P <0.05". The authors should use P ≥ 0.05 instead of P > 0.05. If they simply want to express that there is no statistical difference, the reviewer recommends using NS (not significant). Otherwise, each figure will be confused. In other articles, the P value is expressed to the second decimal place even if there is no significant difference.

Reviewer #2: The authors have adequately addressed my comments raised in a previous round of review. I believe that this manuscript is now acceptable for publication.

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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

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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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Angiotensin-(3–4) normalizes the elevated arterial blood pressure and abnormal Na+/energy handling associated with chronic undernutrition by counteracting the effects mediated by type 1 angiotensin II receptors

PONE-D-22-09686R2

Dear Dr. Vieyra

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Xianwu Cheng, M.D., Ph.D., FAHA

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All concerns have been addressed by the authors.

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: All comments have been addressed

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: I Don't Know

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: The revised version is corrected as the reviewer point outed.　The figures are much easier understand.

Reviewer #2: All the concerns have been fully addressed. I think the current version can be accepttable for publication now.

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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

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