PONE-D-19-35917

Renoprotective effects of paramylon, a β-1,3-D-Glucan isolated from Euglena gracilis Z in a rodent model of chronic kidney disease

PLOS ONE

Dear Dr. Nagayama,

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.

The manuscript was revised and we appreciate if the authors could reply to all issues discriminated below.

We would appreciate receiving your revised manuscript by Mar 29 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Niels Olsen Saraiva Câmara, M.D, PhD

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Competing Interests/Financial Disclosure* (delete as necessary) section:

"I have read the journal's policy and the authors of this manuscript have the following competing interests: Ms. Ayaka Nakashima and Dr. Kengo Suzuki are employees of euglena Co., Ltd."

We note that one or more of the authors are employed by a commercial company: euglena Co., Ltd.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

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: 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: The manuscript submitted by Nagayama et al describes the renoprotective effects of paromylon, a Beta-1,3-D-Glucan in a rodent model of chronic kidney disease. The treatment showed improvement of the kidney function and decreased accumulation of TCA cycle metabolites and uremic toxins.

Major comments:

-Figure 2 a, b and C: use arrows to show where the injury/damage is on the pictures for glomerulosclerosis, tubular injury, glomerular hypertrophy.

- Improve the images quality. Show the bar graphs with dots for each mouse.

- Figure 1 can be joint to Figure 2, as well as the results sections linked to both figures.

-Figure 3: Improve the images quality. Use arrows to show where the injury/damage is.

Don't use the word tendency to describe the non-significant differences in the results section. Use dots to represent each mouse on the graphs (dot plot).

- Figure 4: It would be nice to see MMP9, IL10 and ROS levels in the kidneys too. It would be interesting to evaluate if there is modulation of the macrophage profile induced by paromylon.

-Figure 5: The treatment decreases the accumulation of TCA cycle metabolites. Improve the discussion about these data. Correlate to improvement of inflammatory parameters such as IL1b.

Improve the quality of the graphs. An alternative to better show this data is to build a heatmap.

The results section “Identification of some uremic toxins using CE-TOFMS analysis” needs to be improved. The title could be changed to something like: Accumulation of TCA cycle metabolites and uremic toxins are decreased in the serum by paromylon treatment.

This section should be better described because these results fall from the sky, without any introduction about why to look at this (TCA cycle etc).

Figure 6: Since Beta glucans are found in fungi species, would you consider changes in fungi species in the microbiome induced by the treatment?

Minor Comments:

-The word “tendency” should be replaced.

-Improve the quality of all images and include the scale bar.

Reviewer #2: This is an interesting manuscript which authors indicating paramylon reduces renal tissue damage and inflammation, however, paramylon has no effect on the intestinal microbiota. However, several points to be revised in the present manuscript to be accept in Plos One, described as follow.

Introduction:

- Must be revised and include an information about the impact of altered intestinal microbiota on CKD progression and the beneficial of dietary fibers in this context.

- Must be cited the original studies about the biological effects of dietary B-glucans described in lines 58-60.

- After citing once “Euglena gracilis Z”, abreviate as “E. gracilis Z”, as described in the others manuscript’s sections.

- Instead of use “CKD rats”, substitute for “CKD rat model”.

Results:

- Revised the use of the “on the other hand” in description of results.

- The scale bars must be insert in the images.

- In the figure 4, identify de graphics with letters (A-F), the control group was not demonstrated in this figure.

- Attenuate the phrase described in lines 306-310. The authors just analyzed gene expression.

- The authors reported no differences in bacteria genera or clusters in microbiota. Maybe the differences could be observed among the species, instead of genera or clusters. Metagenomic assay will be very much appreciated to verify this point.

- In the literature has been hypothesized that increased gut permeability contributes to inflammation in patients with advanced CKD. Was the gut tissue analyzed in the present study? Sometimes the amelioration induced by paramylon could be related to improve de mucosal permeability, and it could be discussed.

Discussion

-The results are very poor discussed. The authors described results in this section instead of discuss them. The discussion must be revised and intersect the findings with data from literature.

-The limitations of techniques used to characterize intestinal microbiome must be discussed. The interface between gut microbiota and CKD must be explored.

In line 452 - “It is unknown whether 5-hydroxylysine, argininosuccinic acid,

N6-methyllysine, N8-acetylspermidine, O-acetylhomoserine, and

galactosylhydroxylysine are uremic toxins”. Were these compounds described in CKD patients or another rodent model of CKD? What the relevance these compounds whether they are unknown their classification as uremic toxins?

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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.

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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-19-35917R1

Renoprotective effects of paramylon, a β-1,3-D-Glucan isolated from Euglena gracilis Z in a rodent model of chronic kidney disease

PLOS ONE

Dear Dr. Nagayama,

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.

Specifically the authors could pay special attention to results section.

Please submit your revised manuscript by Jul 23 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Niels Olsen Saraiva Câmara, M.D, PhD

Academic Editor

PLOS ONE

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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: (No Response)

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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: I Don't Know

**********

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: No

**********

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 authors provided answer for all my points. The quality of this manuscript improved considerably, however in the results section, the results could be more explored and the authors could provide more background on why they performed those experiments, before presenting the results.

Reviewer #2: The findings reported by Nagayama and colleagues are interesting and present potential to be published in Plos One. Although, the authors had accepted all suggestions made by reviewers in the first moment, the article still need to be adequate to formal language, and the revision by a native English speaker is mandatory. In this second revision, the discussion section needs more attention, the authors use inadequate grammar and unnecessary sentences to justify the findings, some examples are listed below.

Discussion

- - Table 2 is suggested as supplemental data;

- - The sentences need to be better connected, using adequate English grammar

- - In line 507, “we investigated whether if paramylon inhibited absorption of uremic toxin with CE-TOFMS analysis”. Review the use of whether/if

- - Some phrases depreciated this work and must be attenuated or excluded: “Unfortunately, we did not get the gut tissue to analyze”; There were several limitations in our study.

- - Dear authors, the discussion must be carefully revised and interconnect the results and then submitted to a native English speaker.

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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.]

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.

Renoprotective effects of paramylon, a β-1,3-D-Glucan isolated from Euglena gracilis Z in a rodent model of chronic kidney disease

PONE-D-19-35917R2

Dear Dr. Nagayama,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Niels Olsen Saraiva Câmara, M.D, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: (No Response)

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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: I Don't Know

**********

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 authors have addressed my concerns. The results section was significantly improved to better explain the findings.

Reviewer #2: (No Response)

**********

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