Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients

Nobuhiko Miyatake; Hiroki Adachi; Kanae Nomura-Nakayama; Keiichiro Okada; Kazuaki Okino; Norifumi Hayashi; Keiji Fujimoto; Kengo Furuichi; Hitoshi Yokoyama

doi:10.1371/journal.pone.0226526

Peer Review History

Original SubmissionJuly 23, 2019
3 Sep 2019 Decision Letter - Tatsuo Shimosawa, Editor PONE-D-19-20735 Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients PLOS ONE Dear Dr. Yokoyama, 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. Sorry for delay of review processes. Two experts raised several serious concerns to draw your conclusion. Please revise the manuscript following their comments. We would appreciate receiving your revised manuscript by Oct 18 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. 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, Tatsuo Shimosawa, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. 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/10.1371/journal.pone.0195066 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. 3. Please include a separate caption for each figure in your manuscript. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: Yes Reviewer #2: Yes ****** 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 ****** 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: This manuscript entitled “Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients” written by Miyatake et al showed that the change in the serum CTRP9 concentration was positively correlated with the change in the serum HMW-ADPN concentration, and that multiple regression analysis showed that the change in the serum CTRP9 concentration was negatively associated with the change in ACAI. These findings are potentially interesting and useful data to understand the increased development of atherosclerotic lesions in renal allograft recipients. However, their data do not have the important confounding factors, such as high sensitive CRP or TNF-α. In addition, immuno-histological staining of Adipo R1 and R2 is quite poor images. 1. As the authors mentioned in Discussion, the concentration of CTRP9 and HMW-ADPN is strongly associated with the inflammatory state. Thus, the authors should measure some inflammatory mediators like high sensitive CRP, TNF-α, IL-6. 2. In Figure 5, the staining of Adipo R1 and R2 looks non-specific. These data do not show the proof of co-localization between CTRP9 and Adipo R1. The authors should exhibit more clear stain. Or, Figure 5 should be removed. Reviewer #2: Novelty: The manuscript is the 1st report on the negative correlation of circulation CTRP9 and aortic calcification in renal transplantation patients. Major issues: 1. Renal allograft IS (co-location of CTRP9 and AdipoR1) was not able to lead the conclusion ‘CTRP9 and HMW-ADPN were suggested to prevent the progression of aortic calcification through AdipoR1.’ Because chronic rejection implicates the allograft vessels directly. To address this concern, animal allogeneic solid organ transplantation model (e.g. heart, kidney or liver) could be set and non-relevant artery (abdominal artery) should be stained for co-location to exclude the direct allorejection background. 2. What’s the relationship of concentration of CTRP9 (and HMW-ADPN) with ACAI at endpoint (2016)? 3. How were the “concentration changes” calculated? This should be described in “Method” section. ****** 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. https://doi.org/10.1371/journal.pone.0226526.r001
Revision 1
12 Nov 2019 Author Response Dear to Editor, Thank you very much for your thoughtful comments. We would like to re–submit the revised manuscript entitled “Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients’’. We are most grateful to you and the reviewers for the helpful comments on the original version of our manuscript. We have taken all these comments into account and submit. According to your reviewer’s comments, we improved our manuscripts as below. The reviewer-1 comments: This manuscript entitled　“Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients” written by Miyatake et al showed that the change in the serum CTRP9 concentration was positively correlated with the change in the serum HMW-ADPN concentration, and that multiple regression analysis showed that the change in the serum CTRP9 concentration was negatively associated with the change in ACAI. These findings are potentially interesting and useful data to understand the increased development of atherosclerotic　 lesions in renal allograft recipients. However, their data do not have the important confounding factors, such as high sensitive CRP or TNF��. In addition, immuno-histological staining of Adipo R1 and R2 is quite poor images. Comment #1: As the authors mentioned in Discussion, the concentration of CTRP9 and HMW-ADPN is strongly associated with the inflammatory state. Thus, the authors should measure some inflammatory mediators like high-sensitive CRP, TNF��, IL-6. Response: We think it difficult to measure the mediators such as high-sensitivity CRP, TNF-α, and IL-6, as you pointed out. One reason is that we thought they were not significantly different because we measured them in a stable state, and another reason is the limited amount of sample needed for measurement. Comment #2: In Figure 5, the staining of Adipo R1 and R2 looks non-specific. These data do not show the proof of co-localization between CTRP9 and Adipo R1. The authors should exhibit more clear stain. Or, Figure 5 should be removed. Response: We re-stained in another case for Adipo R1 and R2 IS (immunofluorescent staining), to confirm more specific staining. Hence, the conditions of reagents used for staining have been changed. As a result, more specific staining was obtained for CTRP9 and AdipoR1. A clearer colocalization was confirmed. We can send the newly obtained image in a manuscript here. The reviewer-2 comments: The manuscript is the 1st report on the negative correlation of circulation CTRP9 and aortic calcification in renal transplantation patients. Comment #1: Renal allograft IS (co-location of CTRP9 and AdipoR1) was not able to lead the conclusion ‘CTRP9 and HMW-ADPN were suggested to prevent the progression of aortic calcification through AdipoR1. Because chronic rejection implicates the allograft vessels directly. To address this concern, animal allogeneic solid organ transplantation model (e.g. heart, kidney or liver) could be set and non-relevant artery (abdominal artery) should be stained for co-location to exclude the direct allorejection background. Response: In this study, the proof of CTRP9 and AdipoR1 collocation in renal allograft IS in this study was aimed to show that the CTRP9 expression site coincides with the localization of AdipoR1. As you pointed out, we think that this IS alone cannot prove the process of suppressing vascular calcification. In addition, animal experiments of organ transplantation models, such as your proposal, will be difficult to carry out at our facility. Comment #2: What’s the relationship of concentration of CTRP9 (and HMW-ADPN) with ACAI at endpoint (2016)? Response: We couldn’t confirm significant correlation CTRP9 and HMW-ADPN concentrations with concurrent ACAI at the 2016 endpoint. Comment #3: How were the “concentration changes” calculated? This should be described in “Method” section. Response: We used the calculation method of concentration change is shown below. Change in CTRP9= {(serum CTRP9 level in 2008)- (serum CTRP9 level in 2016)}/8 Change in HMW ADPN= {(serum HMW-ADPN level in 2008)- (serum HMW-ADPN level in 2016)}/8 Change in ACAI= {(ACAI in 2008)- (ACAIin 2016)}/8 The method for calculating the concentration change is shown in the Method section. We will continue to make corrections centered on the above points and send the revised manuscript by the due date. We thank for your advices. Nobuhiko Miyatake, MD C/O Hitoshi Yokoyama, MD, PhD Kanazawa Medical University School of Medicine Department of Nephrology 1-1 Daigaku, Uchinada, Ishikawa 920-0293, JAPAN. Tel: +81-76-218-8166 FAX: +81-76-286-2786 E-mail: nobuhiko@kanazawa-med.ac.jp Attachments Attachment Submitted filename: response to reviewers .docx https://doi.org/10.1371/journal.pone.0226526.r002
21 Nov 2019 Decision Letter - Tatsuo Shimosawa, Editor PONE-D-19-20735R1 Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients PLOS ONE Dear Dr. Yokoyama, 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 authors could not measure inflammatory markers because they did not plan it and store samples. It is a limitations of this study. Authors stated that under stable condition, those markers does not differ. If so, authors should show scientific proof. We would appreciate receiving your revised manuscript by Jan 05 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, Tatsuo Shimosawa, M.D., 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 ******** 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: Partly 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: (No Response) 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: In my previous comment in the authors’ first submission, inflammatory mediators like hs-CRP, TNF-α or IL6 should be measured because the concentration of CTRP9 and HMW-ADPN is strongly associated with the inflammatory state. However, he authors has not measured at all. In addition, re-staining of Figure 5 has not successfully shown the colocalization between CTRP9 and AdipoR1. In total, the authors has not appropriately revised the manuscript at all. Reviewer #2: Most of my comments were well addressed, although some of the suggested experimental methods was not able to performed technically. In the revision, the fonts in line 183-188 are significantly larger than others. Please format. Furthermore, some of the Figure legends were inserted into the ‘Result’ section, which confused me. The manuscript is suitable for publication if the above issues are corrected. ****** 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. https://doi.org/10.1371/journal.pone.0226526.r003
Revision 2
28 Nov 2019 Author Response Date: November 26, 2019 Editor office “PLOS ONE” Dear to Editor, Thank you very much for your thoughtful comments. We would like to re–submit the revised manuscript entitled “Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients’’. We are most grateful to you and the reviewers for the helpful comments on the original version of our manuscript. We have taken all these comments into account and submit. According to your reviewer’s comments, we improved our manuscripts as below. Comment #1: Thank you very much for your thoughtful comment. Unfortunately, this study was a retrospective study, then the collected samples were insufficient in some cases. Therefore, it was difficult to measure additionally and to compare with other suggested molecules such as high sensitivity CRP and TNF-α. We added the following sentence in limitation; 3) not using high accurate inflammatory markers such as sensitive CRP or TNF��. However, the concentration of CTRP9 or HMW-ADPN is also strongly associated with the inflammatory state as we mentioned in Discussion. We hope our revision certified your comment. We thank you again for your advice. Nobuhiko Miyatake, MD C/O Hitoshi Yokoyama, MD, PhD Kanazawa Medical University School of Medicine Department of Nephrology 1-1 Daigaku, Uchinada, Ishikawa 920-0293, JAPAN. Tel: +81-76-218-8166 FAX: +81-76-286-2786 E-mail: nobuhiko@kanazawa-med.ac.jp Attachments Attachment Submitted filename: response to reviewers 20191126.docx https://doi.org/10.1371/journal.pone.0226526.r004
3 Dec 2019 Decision Letter - Tatsuo Shimosawa, Editor Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients PONE-D-19-20735R2 Dear Dr. Yokoyama, 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. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Tatsuo Shimosawa, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0226526.r005
Formally Accepted
10 Jan 2020 Acceptance Letter - Tatsuo Shimosawa, Editor PONE-D-19-20735R2 Circulating CTRP9 correlates with the prevention of aortic calcification in renal allograft recipients Dear Dr. Yokoyama: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Tatsuo Shimosawa Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226526.r006

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