Klotho deficiency intensifies hypoxia-induced expression of IFN-α/β through upregulation of RIG-I in kidneys

Asako Urabe; Shigehiro Doi; Ayumu Nakashima; Takeshi Ike; Kenichi Morii; Kensuke Sasaki; Toshiki Doi; Koji Arihiro; Takao Masaki

doi:10.1371/journal.pone.0258856

Peer Review History

Original SubmissionApril 22, 2021
3 Jun 2021 Decision Letter - Niels Olsen Saraiva Câmara, Editor PONE-D-21-13393 Klotho deficiency intensifies hypoxia-induced expression of IFN- α / β through upregulation of RIG-I PLOS ONE Dear Dr. Doi, 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 has been revised and we suggest the authors to reply point-by-point the comments raised by the referee. Please submit your revised manuscript by Jul 18 2021 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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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 ******** 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: The authors performed in vitro and in vivo studies and used human biopsies to assess the effect of hypoxia in RIG-mediated IFNα/β expression in kidneys. They demonstrated that RIG-I is responsible for the hypoxia induced upregulation of IFN-α/β and that klotho deficiency intensifies this process. This is a concise, well thought out study. Although interesting, some points represent flaws, and need to be addressed. Major - It is not clear what the research hypothesis is. Please make it clear in the Introduction section. - The authors should describe in Materials and Methods how they kept the NRK-52E cells under hypoxic conditions. - Figure 1: How was the expression of RIG-I after 60, 90, and 120 min of hypoxic stimulation? Was this parameter increased in a time-dependent manner? - Please provide evidence that cells and kidneys were under hypoxia. The authors should perform an assay with hypoxia markers (e.g. pimonidazole) or evaluate the expression of hypoxia-related genes (e.g. EPO, VEGF…). - Hypoxia promotes activation of HIF-1a, which regulates the expression of several genes. Is there a role for HIF-1a in the regulation of renal klotho/RIG-I/IFN in this study? - Authors should consider using a klotho inhibitor to check the expression of RIG-I and IFN in NRK-52E cells under hypoxia. - In the Conclusion section, the Authors mentioned that “Klotho expression reportedly decreases with aging and the progression of CKD [41-42], suggesting that hypoxia-induced RIG-I upregulation likely occurs in the elderly and CKD patients”. However, the present manuscript does not provide data to support such conclusion. Minor - The short tittle does not match the tittle. Please correct it. - Figure 3A: Please include the name of the groups. Reviewer #2: The manuscript by Asako Urabe and colleagues tries to propose Klotho downregulation increases hypoxia-induced expression of IFN- α / β by overexpression of Retinoic acid inducible gene I (RIG-I). Authors showed that hypoxia induces the expression of IFN-α/β through the upregulation of RIG-I and suggesting that hypoxia-induced RIG-I upregulation likely occurs in the elderly and CKD patients. 1. In my opinion, in title and keywords would be better to add kidney. 2. Microscopic magnification as a bar or numerical value must be indicated in all Figures and Legends. It would also be helpful if the authors used arrows to point out the changes they are describing in the figure legend and text, for example the expression and localization in RIG-I in WT and Kl-/- mice. 3. Please emphasize on the clinical importance of your findings. Please discuss how your results may translate into clinical practice. 4. The author should state the dose of anesthesia. 5. In M&M, several sentences need specific references e.g. Transfection of RIG-I siRNA part. Please add ****** 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.] 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. https://doi.org/10.1371/journal.pone.0258856.r001
Revision 1
28 Aug 2021 Author Response Responses to Reviewer #1 Thank you for your thorough review and constructive feedback on our manuscript. We have revised the manuscript in accordance with your comments. Our responses to each comment are shown below: 1. It is not clear what the research hypothesis is. Please make it clear in the Introduction section. Response to comment 1 Following your suggestion, we rewrote the Introduction section. 2. The authors should describe in Materials and Methods how they kept the NRK-52E cells under hypoxic conditions. Response to comment 2 In this study, the NRK-52E cells were seeded into 10 cm culture dishes. After growing to subconfluence, the cells were incubated in a 1% O2 Modular Incubator Chamber (MIC 101; Billups-Rothenberg, San Diego, CA) for 30, 60, 90, and 120 min. We added a description of this in the Materials and Methods section. 3. Figure 1: How was the expression of RIG-I after 60, 90, and 120 min of hypoxic stimulation? Was this parameter increased in a time-dependent manner? Response to comment 3 The expression of RIG-I peaked at 30 min and then gradually decreased under hypoxic conditions. We replaced Figure 1A to show this. 4. Please provide evidence that cells and kidneys were under hypoxia. The authors should perform an assay with hypoxia markers (e.g. pimonidazole) or evaluate the expression of hypoxia-related genes (e.g. EPO, VEGF…). Response to comment 4 We performed western blotting for HIF-1α in the NRK-52E cells and EPO in the hypoxic kidneys to provide evidence that the cells and kidneys were under hypoxia. HIF-1α expression increased during 120 min of hypoxic stimulation in the NRK-52E cells, while EPO expression was upregulated at 10 min of ischemia in both WT and klotho-knockout mice. We added these findings in the Supporting Information. 5. Hypoxia promotes activation of HIF-1a, which regulates the expression of several genes. Is there a role for HIF-1a in the regulation of renal klotho/RIG-I/IFN in this study? Response to comment 5 Previous study demonstrated that RIG-I is a downstream effector of HIF-1α. However, HIF-1α expression gradually increased during 120 min of ischemia, while RIG-I expression peaked at 30 min. Moreover, siRNA for HIF-1a did not suppress the hypoxia-induced upregulation of RIG-I (data not shown). Thus, we could not identify that the upregulation of HIF-1α is responsible for RIG expression. 6. Authors should consider using a klotho inhibitor to check the expression of RIG-I and IFN in NRK-52E cells under hypoxia. Response to comment 6 Unfortunately, klotho is not expressed in NRK-52E cells. Therefore, we could not perform an in vitro experiment using a klotho inhibitor. However, in vivo study clearly showed the effect of klotho on the expression of RIG-I and IFN-α/β under hypoxia. 7. In the Conclusion section, the Authors mentioned that “Klotho expression reportedly decreases with aging and the progression of CKD [41-42], suggesting that hypoxia-induced RIG-I upregulation likely occurs in the elderly and CKD patients”. However, the present manuscript does not provide data to support such conclusion. Response to comment 7 We previously showed that klotho expression is associated with aging and the progression of CKD. Another study also reported this. However, to avoid confusion, we rewrote the Discussion section of the manuscript. 8. The short tittle does not match the tittle. Please correct it. Response to comment 8 Following your suggestion, we corrected the short title. 9. Figure 3A: Please include the name of the groups. Response to comment 9 Following your suggestion, we included the names of the groups. Responses to Reviewer #2 Thank you for your thorough review and constructive feedback on our manuscript. We have revised the manuscript in accordance with your comments. Our responses to each comment are shown below: 1. In my opinion, in title and keywords would be better to add kidney. Response to comment 1 Following your suggestion, we added “kidney” to the keywords and title. 2. Microscopic magnification as a bar or numerical value must be indicated in all Figures and Legends. It would also be helpful if the authors used arrows to point out the changes they are describing in the figure legend and text, for example the expression and localization in RIG-I in WT and Kl-/- mice. Response to comment 2 Following your suggestion, we added a bar to show the microscopic magnification in all figures and legend. We also added arrows to point out the changes. 3. Please emphasize on the clinical importance of your findings. Please discuss how your results may translate into clinical practice. Response to comment 3 Following your suggestion, we rewrote the Discussion section of the manuscript. 4. The author should state the dose of anesthesia. Response to comment 4 We added the dose of anesthesia in the Methods section. 5. In M&M, several sentences need specific references e.g. Transfection of RIG-I siRNA part. Please add Response to comment 5 RIG-I siRNA was used in accordance with the manufacturer’s instructions. We also added a reference on the anesthesia in the Material and Methods section. Thank you for pointing out some issues with our manuscript. We have revised the manuscript in accordance with your comments. Our responses to each comment are shown below: 1.Please amend your Methods section to state the method of euthanasia/sacrifice of the rats. Response to comment 1 The method of euthanasia/sacrifice was cardiac puncture. Following your suggestion, we have added this to the Methods section. 2.We now require authors to provide the original unadjusted and uncropped images for any blot or gel data reported in PLOS ONE submissions. In our internal checks for your submission, we noted that you did not provide original raw image files supporting blot/gel data in response to our previous request. Please provide the unadjusted and uncropped images underlying all blot and gel figures at this time; see the above URL for instructions on how the raw blot/gel image data should be prepared and submitted. Response to comment 2 Following your suggestion, we have provided the original unadjusted and uncropped images in the Supplemental Information. 3.We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: 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. Response to comment 3 As you have pointed out, some of the Methods section overlapped with our previous publication. Following your suggestion, we have rewritten the Methods section to resolve this. Attachments Attachment Submitted filename: renamed_e4e28.docx https://doi.org/10.1371/journal.pone.0258856.r002
7 Oct 2021 Decision Letter - Niels Olsen Saraiva Câmara, Editor Klotho deficiency intensifies hypoxia-induced expression of IFN- α / β through upregulation of RIG-I in kidneys PONE-D-21-13393R1 Dear Dr. Doi, 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) ******** 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: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) 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: Yes:** Hassan Askari https://doi.org/10.1371/journal.pone.0258856.r003
Formally Accepted
11 Oct 2021 Acceptance Letter - Niels Olsen Saraiva Câmara, Editor PONE-D-21-13393R1 Klotho deficiency intensifies hypoxia-induced expression of IFN-α/β through upregulation of RIG-I in kidneys Dear Dr. Doi: 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 Prof. Niels Olsen Saraiva Câmara Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258856.r004

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