PONE-D-24-14449Upregulation of Metrnl improves diabetic kidney disease by inhibiting the TGF-β1/Smads signaling pathway: A potential therapeutic targetPLOS ONE

Dear Dr. chen,

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.

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Reviewer #1: Lin et al. investigated the role of Metrnl in diabetic kidney disease (DKD) using db/db mice and explored the possible mechanism involving the TGF-β1/Smad signaling pathway. They found that overexpression of Metrnl in db/db mice improved renal function and reduced the expression of inflammatory factors such as TNF-α, TGF-β1, and α-SMA. They also found that Pearson correlation analysis revealed a negative association between UACR and Metrnl, and a positive correlation between UACR and TGF-β1 in db/db mice. They concluded that upregulation of renal Metrnl expression may attenuate renal injury by modulating the TGF-β1/Smad signaling pathway in DKD. This study may shed light on potential therapeutic targets for the treatment of DKD in type 2 diabetes, but some concerns need to be addressed.

In 2.1, the authors should provide details of the adenoviral vectors used in this study. Are the adenoviral vectors designed to overexpress Metrnl in db/db mice systemically or in a tissue-specific manner driven by the promoters and enhancers?

In 2.4, the authors describe that the DKD+NS group received adenovirus dilutions in saline. The reviewer believes that only saline was injected in these control groups and this point should be verified. The authors also need to specify whether the NC group received the control adenovirus vector or saline.

In Figures 1C-D, the authors showed that overexpression of Metrnl in db/db mice resulted in a reduction of FBS and UACR. According to the previous paper cited as reference 13, BMI, HbA1c and ACR were reported to be associated with serum Metrnl in patients with type 2 diabetes mellitus. Why do you think that the db/db mice overexpressing Metrnl did not lose body weight as shown in Figure 1B? The reviewer also thinks that it would be better to show other biochemical parameters such as BUN, TG, HDL-C in db/db mice overexpressing Metrnl.

In Figures 3E and 4F, the authors evaluated changes in the expression of Metrnl, TGF-beta, TNF-alpha and alpha-SMA proteins expressed as a relative positive staining index. Authors should provide the procedures for their semi-quantitative analyses in the Materials and Methods section.

In Figure 4A-B, the thylakoid cells and inflammatory cells were difficult to see. It would be better to make the figures easier to understand, for example, by inserting arrows or asterisks.

The authors describe that glomerular size, thylakoid cell proliferation, and the number of inflammatory cells were reduced in db/db mice overexpressing Metrnl. The authors should provide semi-quantitative analysis data to support those descriptions.

The TEM images shown in Figures 4C-D are unclear and should be replaced.

In their interpretation of Figure 5, the authors use the term "significant" to describe pathological changes in db/db mice overexpressing Metrnl. However, the reviewers believe that the authors' descriptions are inappropriate because they have not provided semi-quantitative analysis data.

The results of TNF-alpha immunostaining shown in Figure 6F are unclear. They should be replaced.

In Figure S1, the authors describe that Mtrnl may attenuate the TGF-β/Smad signaling pathway in renal tissue and prevent fibrosis in glomeruli and tubules. However, in Figure 6, TGF-β protein was mainly detected in glomeruli and α-SMA protein was mainly detected in tubular lesions. What mechanisms do you think that Mtrnl-mediated suppression of TGF-β in glomeruli would prevent fibrosis in glomeruli and tubules?

In lines 443-444, the description should be corrected.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data. 

==============================

Please submit your revised manuscript by Jul 24 2024 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Md Shaifur Rahman, Ph.D

Academic Editor

PLOS ONE

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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

**********

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

Reviewer #1: Yes

**********

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

**********

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

**********

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: Lin et al. investigated the role of Metrnl in diabetic kidney disease (DKD) using db/db mice and explored the possible mechanism involving the TGF-β1/Smad signaling pathway. They found that overexpression of Metrnl in db/db mice improved renal function and reduced the expression of inflammatory factors such as TNF-α, TGF-β1, and α-SMA. They also found that Pearson correlation analysis revealed a negative association between UACR and Metrnl, and a positive correlation between UACR and TGF-β1 in db/db mice. They concluded that upregulation of renal Metrnl expression may attenuate renal injury by modulating the TGF-β1/Smad signaling pathway in DKD. This study may shed light on potential therapeutic targets for the treatment of DKD in type 2 diabetes, but some concerns need to be addressed.

In 2.1, the authors should provide details of the adenoviral vectors used in this study. Are the adenoviral vectors designed to overexpress Metrnl in db/db mice systemically or in a tissue-specific manner driven by the promoters and enhancers?

In 2.4, the authors describe that the DKD+NS group received adenovirus dilutions in saline. The reviewer believes that only saline was injected in these control groups and this point should be verified. The authors also need to specify whether the NC group received the control adenovirus vector or saline.

In Figures 1C-D, the authors showed that overexpression of Metrnl in db/db mice resulted in a reduction of FBS and UACR. According to the previous paper cited as reference 13, BMI, HbA1c and ACR were reported to be associated with serum Metrnl in patients with type 2 diabetes mellitus. Why do you think that the db/db mice overexpressing Metrnl did not lose body weight as shown in Figure 1B? The reviewer also thinks that it would be better to show other biochemical parameters such as BUN, TG, HDL-C in db/db mice overexpressing Metrnl.

In Figures 3E and 4F, the authors evaluated changes in the expression of Metrnl, TGF-beta, TNF-alpha and alpha-SMA proteins expressed as a relative positive staining index. Authors should provide the procedures for their semi-quantitative analyses in the Materials and Methods section.

In Figure 4A-B, the thylakoid cells and inflammatory cells were difficult to see. It would be better to make the figures easier to understand, for example, by inserting arrows or asterisks.

The authors describe that glomerular size, thylakoid cell proliferation, and the number of inflammatory cells were reduced in db/db mice overexpressing Metrnl. The authors should provide semi-quantitative analysis data to support those descriptions.

The TEM images shown in Figures 4C-D are unclear and should be replaced.

In their interpretation of Figure 5, the authors use the term "significant" to describe pathological changes in db/db mice overexpressing Metrnl. However, the reviewers believe that the authors' descriptions are inappropriate because they have not provided semi-quantitative analysis data.

The results of TNF-alpha immunostaining shown in Figure 6F are unclear. They should be replaced.

In Figure S1, the authors describe that Mtrnl may attenuate the TGF-β/Smad signaling pathway in renal tissue and prevent fibrosis in glomeruli and tubules. However, in Figure 6, TGF-β protein was mainly detected in glomeruli and α-SMA protein was mainly detected in tubular lesions. What mechanisms do you think that Mtrnl-mediated suppression of TGF-β in glomeruli would prevent fibrosis in glomeruli and tubules?

In lines 443-444, the description should be corrected.

**********

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

**********

[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.

PONE-D-24-14449R1Upregulation of Metrnl improves diabetic kidney disease by inhibiting the TGF-β1/Smads signaling pathway: A potential therapeutic targetPLOS ONE

Dear Dr. chen,

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.

1. The manuscript would need a partial review of the English language. The presenting content has minor spelling and grammatical errors, especially in the abstract.

2. Some of the sentences have no references, please revise and add their references. For example: "Several studies have examined the association between Metrnl level and adverse diabetic renal events in patients with DKD. These studies found a negative correlation between serum Metrnl concentration and the risk of DKD", Ref?

3. In the purpose section of the abstract, "This study aimed to investigate the role of Metrnl in this model" What do you mean by "this"?

4. It is suggested to add a box of abbreviations before the abstract, many of the abbreviations in the abstract are incomprehensible.

5. The mechanism of action of Metrnl in the control of diabetic kidneys is better depicted in a figure or graphical abstract.

6. Some explanations in the discussion are not needed. Please add the results of previously assessed studies and justify the obtained results. 

In addition to above, could you please clarify how you generated the Supp. fig. 2 (mouse with organ)

Please submit your revised manuscript by Aug 25 2024 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,

Md Shaifur Rahman, Ph.D

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:

Dear Authors,

Please consider the following points raised by reviewer.

1. The manuscript would need a partial review of the English language. The presenting content has minor spelling and grammatical errors, especially in the abstract.

2. Some of the sentences have no references, please revise and add their references. For example: "Several studies have examined the association between Metrnl level and adverse diabetic renal events in patients with DKD. These studies found a negative correlation between serum Metrnl concentration and the risk of DKD", Ref?

3. In the purpose section of the abstract, "This study aimed to investigate the role of Metrnl in this model" What do you mean by "this"?

4. It is suggested to add a box of abbreviations before the abstract, many of the abbreviations in the abstract are incomprehensible.

5. The mechanism of action of Metrnl in the control of diabetic kidneys is better depicted in a figure or graphical abstract.

6. Some explanations in the discussion are not needed. Please add the results of previously assessed studies and justify the obtained results.

In addition to above, could you please clarify how you generated the Supp. fig. 2 (mouse with organ)

Thanks.

[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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

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 #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 #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 #2: 1. The manuscript would need a partial review of the English language. The presenting content has minor spelling and grammatical errors, especially in the abstract.

2. Some of the sentences have no references, please revise and add their references. For example: "Several studies have examined the association between Metrnl level and adverse diabetic renal events in patients with DKD. These studies found a negative correlation between serum Metrnl concentration and the risk of DKD", Ref?

3. In the purpose section of the abstract, "This study aimed to investigate the role of Metrnl in this model" What do you mean by "this"?

4. It is suggested to add a box of abbreviations before the abstract, many of the abbreviations in the abstract are incomprehensible.

5. The mechanism of action of Metrnl in the control of diabetic kidneys is better depicted in a figure or graphical abstract.

6. Some explanations in the discussion are not needed. Please add the results of previously assessed studies and justify the obtained results.

**********

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 #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.

Attachments

Attachment

Submitted filename: Dear respected editor.docx

Upregulation of Metrnl improves diabetic kidney disease by inhibiting the TGF-β1/Smads signaling pathway: A potential therapeutic target

PONE-D-24-14449R2

Dear Dr. Chen,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Md Shaifur Rahman, Ph.D

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 #3: All comments have been addressed

**********

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 #3: Partly

**********

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

Reviewer #3: 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 #3: No

**********

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 #3: 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 #3: Review Comments to the Author:

Title:

Upregulation of Metrnl improves diabetic kidney disease by inhibiting the TGF-β1/Smads signaling pathway: A potential therapeutic target

There are no new comments for authors

Thanks

**********

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 #3: No

**********