Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients

Hege Kampen Pihlstrøm; Thor Ueland; Annika E. Michelsen; Pål Aukrust; Franscesca Gatti; Clara Hammarström; Monika Kasprzycka; Junbai Wang; Guttorm Haraldsen; Geir Mjøen; Dag Olav Dahle; Karsten Midtvedt; Ivar Anders Eide; Anders Hartmann; Hallvard Holdaas

doi:10.1371/journal.pone.0243759

Peer Review History

Original SubmissionJune 16, 2020
7 Sep 2020 Decision Letter - Frank JMF Dor, Editor PONE-D-20-18424 Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients PLOS ONE Dear Dr. Pihlstrøm, 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. ============================== ACADEMIC EDITOR: Generally well-executed study albeit with quite a few limitations (acknowledged by authors), but seems to have merit if reviewers' concerns can be adequately addressed by authors. ============================== Please submit your revised manuscript by Oct 22 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. 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PLOS ONE requires that methods are described in enough detail to allow suitably skilled investigators to fully replicate and evaluate the study. Please provide more details on the methods and sample types used to quantify the inflammatory markers in your study (i.re, RNA quantification and ELISA experiments) and all sources and catalog numbers of reagents and ELISA kits used to measure them. Please also clarify which tests were run at the one year visit. If a questionnaire was used during the visit, ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. 3. We note that you have indicated that data from this study are available upon request. 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We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: The authors examined markers of inflammation in patients with a recent renal transplant in a RCT involving the use of paricalcitol to see if paricalcitol would aid in the reduction of inflammation. The study was designed to investigate a different primary outcome (which the authors have published separately). Here, they analysis the results of cytokine analysis as well as microarray analysis. All statistical analyses seem appropriately performed. Table 1 & 2 are nicely put together, with easily identifiable information provided in the table description/legend. It is clear what data is normally distributed and what was not. It owuld be nice when the authors present a result in the text with a p-value to also include which type of statistical test was run to obtain this p-value. This will make the manuscript even more reproducible. Figure 2 in the version that printed for this reviewer does not contain the gene name information. The powerpoint linked in the online version, does. It should be made certain that the gene information appears in the final version, as this is important information to convey to the reader. The authors did a nice job of describing study limitations. Reviewer #2: Generally well written and adequate interpretation of a study with many limitations as the authors have pointed out. I note the following major points - 1. The authors are measuring inflammatory markers post transplantation and they could have looked at the effect of paricalcitol on inflammatory metabolites such as metabolites eg TMAO, p-cresyl sulphate (PCS), p-cresyl glucuronide (PCG), indoxyl sulphate (IS). 2. There is quite a bit of vague terminology e.g. in the 1st paragraph - "inflammatory imbalance in the immune system". What does this actually mean? "interleukin-6 (IL-6) and its major product, CRP" - CRP is not a product of Il-6, instead Il-6 can control hepatic CRP generation. 3. Is the statistical analysis for data in table 2 performed with ANCOVA? This would be more appropriate than multiple t-tets. How did the authors control for multiple statistical testing; could they have applied a Bonferroni correction? 4. Soluble CD25 is not a well validated marker for T cell activation. I would remove this data unless they can demonstrate CD25 expression on isolated T cells by FACS. 5. In discussion, "evidence nevertheless seems too inconsistent to motivate the routine use of VDRA to reduce inflammation or improve vascular health in the transplant population". How could the current study be adapted to answer this? ****** 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. 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Attachments Attachment Submitted filename: PONE-D-20-18424_reviewer.pdf https://doi.org/10.1371/journal.pone.0243759.r001
Revision 1
28 Oct 2020 Author Response A: 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. ANSWER: We have gone through the requirements once more and hopefully corrected any examples of lacking/incorrect information. 2. PLOS ONE requires that methods are described in enough detail to allow suitably skilled investigators to fully replicate and evaluate the study. Please provide more details on the methods and sample types used to quantify the inflammatory markers in your study (i.re, RNA quantification and ELISA experiments) and all sources and catalog numbers of reagents and ELISA kits used to measure them. ANSWER: Thank you for pointing out this requirement! We have now included a more elaborate description of the enzyme immunoassay in the methods section of the manuscript As for the gene expression analysis, an elaborate method description is to be found in the main study publication (ref 29)., which we have cited in the current manuscript. Do you still want it repeated explicitly in the text? Please also clarify which tests were run at the one year visit. If a questionnaire was used during the visit, ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. ANSWER: The original publication listed as ref 29 (Pihlstrom et al, Transplant International 2017, PMID: 28436117) describes in detail what tests were performed at the 1-year visit. On your request we have included a sentence briefly summing up these investigations in the “study design” chapter. For the current analyses, the only relevant “tests” are the biomarker analyses, which were performed en bloc at our Research Laboratory as described above. No questionnaire was part of the data collection process. 3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. ANSWER: Unfortunately there are ethical and legal restrictions preventing us from uploading data to public repositories or including the full dataset as Supplementary Material. Norwegian kidney transplant recipients receiving their transplant in the time-frame of the current study belong to a relatively small group, and very little personal data would be needed in order to indirectly identify individual study participants. We have been in dialogue with the Data Protection Authority of Oslo University Hospital in this matter, and here is their response: "CONCERNING SHARING OF RESEARCH DATA Pursuant to Regulation (EU) No. 2016/679, General Data Protection Regulation (GDPR) article 37, the designated Data Protection Officer at Oslo University Hospital (OUS) is appointed. The controller and the processor shall ensure that the data protection officer is involved, properly and in a timely manner, in all issues which relate to the protection of personal data, cf. GDPR article 38. According to GDPR article 9, the processing of genetic data or health data shall be prohibited unless the data subject has given an explicit consent to the processing of those personal data for one or more specified purposes. Personal data is defined as any information relating to an identified or identifiable natural person; an identifiable natural person is one who can be identified, directly or indirectly. Consequently, depositing de-identified data in a public, community-supported repository in order to submit an article is not considered compliant with EU and Norwegian law in this matter. In order to comply with the relevant legislations, data would need to be fully anonymized. If required, provisions can be made for the inspection of the data as long as the data is under the hospital's control, hence the Data Controller-responsibility". The implication of the above described regulations is that any reader interested in inspecting the data may prompt this request to the OUS Data Protection Officer, Tor Åsmund Martinsen (personvern@oslo-universitetssykehus.no). The corresponding author, Hege Kampen Pihlstrøm (hegphi@ous-hf.no) should also be contacted. A de-identified study data file may then be made available. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. ANSWER: We appreciate the policy that the findings referred to in the manuscript should be available in more detail than what we have provided. Hence we have chosen to include supplementary tables (1-2) for sensitivity analyses and added them to the submission as Supporting Information Files. Reviewer #1: The authors examined markers of inflammation in patients with a recent renal transplant in a RCT involving the use of paricalcitol to see if paricalcitol would aid in the reduction of inflammation. The study was designed to investigate a different primary outcome (which the authors have published separately). Here, they analysis the results of cytokine analysis as well as microarray analysis. All statistical analyses seem appropriately performed. Table 1 & 2 are nicely put together, with easily identifiable information provided in the table description/legend. It is clear what data is normally distributed and what was not. It would be nice when the authors present a result in the text with a p-value to also include which type of statistical test was run to obtain this p-value. This will make the manuscript even more reproducible. ANSWER: Reference to the tests used has been added in the text! Figure 2 in the version that printed for this reviewer does not contain the gene name information. The powerpoint linked in the online version, does. It should be made certain that the gene information appears in the final version, as this is important information to convey to the reader. ANSWER: We will ensure the final figure 2 contains the gene information! Thank you for pointing out this lapse in the version you received. The figure has also been revised (removal of genes related to CD25 as recommended by reviewer 2), and we have included an annotation in the figure legend for genes in the heat map which are known by different names than the proteins they encode. The authors did a nice job of describing study limitations. Reviewer #2: Generally well written and adequate interpretation of a study with many limitations as the authors have pointed out. I note the following major points - 1. The authors are measuring inflammatory markers post transplantation and they could have looked at the effect of paricalcitol on inflammatory metabolites such as metabolites eg TMAO, p-cresyl sulphate (PCS), p-cresyl glucuronide (PCG), indoxyl sulphate (IS). ANSWER: Thank you for the valuable comment! It would indeed have been interesting to investigate inflammatory metabolites as well as the selected circulating biomarkers. However, we are already close to the limit of parameters possible to investigate in this dataset due to its limited size. Hence we feel the analyses of colonic microbiota-derived uraemic retention solutes would be a great idea for a future study, but unfortunately out of the scope of the current project. A comment has been included in the discussion section, suggesting these inflammatory metabolites as focus for upcoming studies. 2. There is quite a bit of vague terminology e.g. in the 1st paragraph - "inflammatory imbalance in the immune system". What does this actually mean? "interleukin-6 (IL-6) and its major product, CRP" - CRP is not a product of Il-6, instead Il-6 can control hepatic CRP generation. ANSWER: Thank you for pointing out imprecise terminology in the manuscript. We have rephrased some sentences in the introduction section according to your comments. Hopefully they will serve as clarification. 3. Is the statistical analysis for data in table 2 performed with ANCOVA? This would be more appropriate than multiple t-tets. How did the authors control for multiple statistical testing; could they have applied a Bonferroni correction? ANSWER: Table 2 presented results of t-tests. As described in the methods section, ANCOVA was performed as a sensitivity analysis. Editors have made it clear that all data which is part of the study should be presented/available for the reader, hence we have included the ANCOVA analyses in a separate column in table 2. Results are comparable to the t-test results. With reference to the two- tailed P-value of ≤0.05 commonly used as cut-off for statistical significance, a p-value of ≤0,003 (0,05/15) would be needed to demonstrate statistical significance after Bonferroni correction for multiple testing in this study. We believe that we have already made it a focus point in the discussion that the study lacks power to conclude firmly on any anti-inflammatory treatment effects, and that the suggested influence of paricalcitol treatment on OPG should be explored in larger studies. We have now added a sentence acknowledging the issue of multiple testing in the limitations section. 4. Soluble CD25 is not a well validated marker for T cell activation. I would remove this data unless they can demonstrate CD25 expression on isolated T cells by FACS. ANSWER: We have removed the data on CD25 as you suggested. 5. In discussion, "evidence nevertheless seems too inconsistent to motivate the routine use of VDRA to reduce inflammation or improve vascular health in the transplant population". How could the current study be adapted to answer this? ANSWER: To be adequately powered to help us conclude on the potential treatment effect of paricalcitol on inflammation in renal transplant recipients, the study should have been larger, with a longer follow-up time. Being a post-hoc analysis of a study designed to investigate a different primary endpoint, we do not have the option of extending the duration of the study or include more patients. We have tried to underscore these limitations at the end of the discussion section. FINAL REMARKS: One of the coauthors, My H S Svensson, did not feel that she had contributed enough to the study and the manuscript to deserve authorship, and consequently we have removed her from the list of authors. Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS 031020.docx https://doi.org/10.1371/journal.pone.0243759.r002
30 Nov 2020 Decision Letter - Frank JMF Dor, Editor Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients PONE-D-20-18424R1 Dear Dr. Pihlstrøm, 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, Frank JMF Dor, M.D., Ph.D., FEBS, FRCS 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: 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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: (No Response) 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: (No Response) Reviewer #2: All my comments have been addressed and no further comments to add from my side. I accept the revised submission ****** 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 https://doi.org/10.1371/journal.pone.0243759.r003
Formally Accepted
3 Dec 2020 Acceptance Letter - Frank JMF Dor, Editor PONE-D-20-18424R1 Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients Dear Dr. Pihlstrøm: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Frank JMF Dor Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0243759.r004

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