Aspirin mediates protection from diabetic kidney disease by inducing ferroptosis inhibition

Ziyu Wu; Dan Li; Dingyuan Tian; Xuejun Liu; Zhongming Wu

doi:10.1371/journal.pone.0279010

Peer Review History

Original SubmissionDecember 21, 2021
21 Apr 2022 Decision Letter - Aldrin V. Gomes, Editor PONE-D-21-40203Aspirin Mediates Protection from Diabetic Kidney Disease by Inducing ferroptosis InhibitionPLOS ONE Dear Dr. Wu, 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. While both reviewers enjoyed reading the manuscript and found it meaningful, some concerns need to be addressed. These concerns include the detection of mitochondrial specific ROS, the rationale for the amount of aspirin used, poor quality western blot images, and grammatical errors in the manuscript. Please submit your revised manuscript by Jun 05 2022 11:59PM. 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We will update your Data Availability statement to reflect the information you provide in your cover letter [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: I Don't Know ****** 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: No 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 is a detailed research article that unraveled the role of ferroptosis and its inhibition in diabetic kidney disease(DKD) using in-vitro HK-2 cells and male diabetic mice model. Although interesting, informative, and thorough, a number of concerns remain. 1. In figure 4, mitochondrial ROS generation was supposedly accessed via DCHF-DA staining. How true is this? DCHF-DA ( 2′ ,7′ -dichlorofluorescein diacetate) can be used to detect total intracellular ROS but certainly not mitochondrial specific ROS. I would suggest the authors revise this section and other sections where mitochondrial ROS was mentioned. 2. The authors mentioned using Aspirin, a non-specific inhibitor of COX-2 activation, at 400uM in cells and 50mg/kg in diabetic mice, but there was no rationale on why those concentration were chosen in their experiments. I would suggest they provide some pharmacological reference backing the concentration if its a low, medium, or high concentration. 3. The manuscript focused on about ~5 protein (GPX4, SLC7A11, FTH-1, TFR-1, COX2) expression. Were other antioxidant and oxidative stress protein looked into and what was their results? It would make sense to look into other pathways and include in the supplemental in order to inform readers. (Plus supplemental is missing as I wasn't able to locate it while reviewing). 4. Involvement of mitochondria should be looked into. Since mitochondria are major sources of ROS within the cell and vulnerable to oxidative stress it would make more sense to investigate their involvement in this Fe2+ overload model of DKD. I would suggest the authors include mitochondrial ROS/superoxide, morphological changes, mitochondrial permeability transition, and oxphos protein expression if possible. 5. Western blotting images could be improved especially SLC7A11 protein expression. 6. English expression needs major expression. I would suggest that grammatical errors in the manuscript be corrected. Reviewer #2: Reviewers' comments: In the present study, Wang et al. found that ferroptosis in renal tubular cells was involved in the development of diabetic kidney disease, and COX2 is a potential target for ferroptosis. They also demonstrated the beneficial effect of aspirin by inhibiting the diabetes-related ferroptosis. The study is well designed and of promising practical guidance. The manuscript is well written and clearly presented. However, there are some issues that the authors should address and they are as follows: 1. How is the level of 4-HNE in high glucose incubated HK2 cells and together with aspirin? It would be better to have consistent results with the animal model. 2. In Fig 4B, COX2 level is increased with DMSO treatment, is there any explanation for this result? 3. The author showed the high glucose increased the cell death in the manuscript and the beneficial effect of aspirin in the animal model, how is the effect of aspirin to the high glucose induced cell death? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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Revision 1
4 Aug 2022 Author Response 、I ensure that my manuscript meets PLOS ONE's style requirements, including those for file naming. 2、My blot/gel image data are in Supporting Information. 3、I ensure that I have an ORCID iD and that it is validated in Editorial Manager. 4、All mice were euthanized with 60 mg/ml of pentobarbital sodium at the end of the experimental period. 5、I have revised the manuscript to rephrase the duplicated text. 6、All authors have declared that no competing interests that pertain to this work.This information have be included in my cover letter. Reviewer #1 Q1:DCHF-DA ( 2′ ,7′ -dichlorofluorescein diacetate) can be used to detect total intracellular ROS, the expression in the text has been modified by us. Q2:The viability of cells treating with RSL-3 was assessed by CCK8. Our study showed that as RSL-3 concentration increased as it was obviously decreased . When RLS-3 concentration was over 0.1 μM, it decreased obviously. As the HK-2 cells was treated with different concentrations of RSL-3 for 48 h, the cell viability was partially recused by Fer-1 400nm or AS 400um treatment. In Fer-1 group and AS group , cells were respectively cultured in 30 mmol/L glucose medium supplemented with 400nM Fer-1 and 400 μM Aspirin for 48 h . Q3:In this study, we mainly explored the relationship between aspirin and ferroptosis, so we did not examine other antioxidant and oxidative stress protein. Q4:Theultrastructural analysis displayed that the changes of mitochondria morphological in the HK-2 cells cultured with high glucose, containing increased membrane density with shrunken mitochondria and mitochondrial ridge decrease or even vanishing, were alleviated by aspirin. The mitochondrial ridge remains obviously visible , the status of increased membrane density and mitochondrial shrinkage were weakened in AS group (Fig 9L). The research implied that the mitochondrial morphological pathogenesis caused by diabetic ferroptosis could be ameliorated by aspirin. Q5:I have fixed the grammar mistakes in the article. Reviewer #2 Q1：In this study, the end products of lipid peroxidation ,MDA,was detected in the cells and animal. Q2：In Fig 4B, COX2 level is increased with DMSO treatment. Because cells were respectively cultured in 30 mmol/L glucose medium supplemented with 0.1% DMSO for 48 h. Q3: In cells being cultured in high-glucose medium with Aspirin,the protein expression of SLC7A11,GPX4, FTH-1 were obviously upregulated while TFR-1 was decreased compared to cells under high-glucose medium ((Fig 4B), these genes exhibited the parallel mRNA expression (Fig 4C-4F).Aspirin treatment markedly attenuated the MDA level of HG group cell(Fig 4I). The GSH was also obviously decreased and the levels of iron was higher in HG group cells,the changes were ameliorated by aspirin in high-glucose medium (Fig 4H,4J).Moreover, the mRNA levels of KIM-1 and NGAL significantly increased in high glucose-cultured HK-2 cells,and these changes were alleviated by aspirin.(Fig 4K-4L) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0279010.r002
15 Sep 2022 Decision Letter - Aldrin V. Gomes, Editor PONE-D-21-40203R1A spirin Mediates Protection from Diabetic Kidney Disease by Inducing ferroptosis InhibitionPLOS ONE Dear Dr. Wu, 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 article requires some improvements in the abstract and text as suggested by the reviewers. 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 Oct 30 2022 11:59PM. 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Gomes, 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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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: This is an improved version of the detailed research article that unraveled the role of ferroptosis and its inhibition in diabetic kidney disease(DKD) using in-vitro HK-2 cells and male diabetic mice model. The experiments in this manuscript were well executed and the rationale behind the experiments support the hypothesis and final conclusion, Although interesting, informative, and thorough, I would suggest a major revision of the Introduction section as it happens to contain a lot of typographical and grammatical errors that cannot be overlooked. Due to the standard of this journal, I would recommend this manuscript to be accepted only after if the author are able to fix the grammatical errors in the manuscript. Some examples of concerns that needs to be rectified but not limited to: 1. Abstract, line 4 and 5: Ferroptosis is a novelty the term linked to lipid hydroperoxidation, and it takes an important part in the pathogenesis of DKD. Suggestion: It should read "Ferroptosis is a novelty term linked to lipid hydroperoxidation, and it plays an important part/role in the pathogenesis of DKD." 2. "DN models" were mentioned in the manuscript multiple times but I believe it should be DM. Please clarify the meaning of DN. 3. Introduction, line 28: Ferroptosis is a novel nonapoptotic cell death that is discovered iron-dependent form in 2012, induced by a redox imbalance redox homeostasis, which is characterized by detoxify lipid oxidation product and free radicals [11]. 4. "These suggesting that by inhibiting ferroptosis in injury PTECs may become a new treatment for kidney" Suggestion: It should be suggest; ... injured PTECs 5. "COX2 as an cyclooxygenase super-family protein that is an inducible enzyme at low levels in most tissues and significantly increased in the proximal tubule of the injured kidney [25,26]" Suggestion: This statement needs to be changed/reworded 6. Results, 3.1, line 22: "Simultaneously, the data showed that the end products of lipid peroxidation, Micro Malondialdehyde Assay Kit was used to assess the level of Malondialdehyde (MDA), the production of MDA was increased by 50% (Fig 1I) compared with the Ctrl group. Suggestion: This sentence is confusing and would be better rewritten in a succinct manner. 7. "We hypothesized that RSL3 was applied to cause ferroptosis in HK-2 cells in order to further explore the role of COX2 in ferroptosis." This word hypothesized isn't needed in this and might be better in a version similar to "We utilized RSL3 to cause ferroptosis in HK-2 cells in order to further explore the role of COX2 in ferroptosis" 8. Furthermore, the viability of cells treating with RSL-3 was assessed by CCK8. Suggestion: It should read the viability of cells treated with with RSL-3 was assessed by CCK8. 9. Moreover, the lipid peroxidation product accumulation,MDA(Fig 6K) and high the concentrations of iron (Fig 6L) under ferroptosis conditions were also attenuated after Fer-1 or aspirin treatment. The word "high the concentrations of iron" is confusing in this sentence. Please endeavor to check for mistakes in order to allow better comprehension of this article and captivate the readers. Reviewer #2: The author answered most of the questions, but the author didn't address the 4-HNE level in high glucose incubated HK2 cells. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0279010.r003
Revision 2
25 Oct 2022 Author Response Reviewer #1: This article contain some typographical and grammatical errors that cannot be overlooked. Thank you for your kind suggestion.We have described these sentence in clearer manner in the revised manuscript, as follows: 1. Abstract, line 4 and 5: Ferroptosis is a novelty the term linked to lipid hydroperoxidation, and it takes an important part in the pathogenesis of DKD. Reply: Ferroptosis is a novel term linked to lipid hydroperoxidation, and it plays an important role in the pathogenesis of DKD. 2."DN models" were mentioned in the manuscript multiple times but I believe it should be DM. Please clarify the meaning of DN. Reply:Thank you for your nice advice.We have removed the DN models from the article. 3. Introduction, line 28: Ferroptosis is a novel nonapoptotic cell death that is discovered iron-dependent form in 2012, induced by a redox imbalance redox homeostasis, which is characterized by detoxify lipid oxidation product and free radicals [11]. Reply:Ferroptosis is a novel nonapoptotic cell death that was discovered to be iron-dependent in 2012 and induced by redox imbalance redox homeostasis, which is characterized by detoxification of lipid oxidation products and free radicals [11]. 4. "These suggesting that by inhibiting ferroptosis in injury PTECs may become a new treatment for kidney" Reply:These results suggest that inhibiting ferroptosis in injured PTECs may become a new treatment for kidney diseases. 5. "COX2 as an cyclooxygenase super-family protein that is an inducible enzyme at low levels in most tissues and significantly increased in the proximal tubule of the injured kidney [25,26]" Reply:COX2 is a cyclooxygenase superfamily protein that is an inducible enzyme at low levels in most tissues and is significantly increased in the proximal tubule of the injured kidney [25,26]. 6. Results, 3.1, line 22: "Simultaneously, the data showed that the end products of lipid peroxidation, Micro Malondialdehyde Assay Kit was used to assess the level of Malondialdehyde (MDA), the production of MDA was increased by 50% (Fig 1I) compared with the Ctrl group. Reply:Simultaneously, the data showed the end products of lipid peroxidation; a micro malondialdehyde assay kit was used to assess the level of malondialdehyde (MDA), and the production of MDA was increased by 50% (Fig. 1I) compared with that in the Ctrl group. 7."We hypothesized that RSL3 was applied to cause ferroptosis in HK-2 cells in order to further explore the role of COX2 in ferroptosis." Reply:We utilized RSL3 to cause ferroptosis in HK-2 cells in order to further explore the role of COX2 in ferroptosis. 8. Furthermore, the viability of cells treating with RSL-3 was assessed by CCK8. Reply:Furthermore, the viability of cells treated with RSL-3 was assessed by CCK8 assay. 9. Moreover, the lipid peroxidation product accumulation,MDA(Fig 6K) and high the concentrations of iron (Fig 6L) under ferroptosis conditions were also attenuated after Fer-1 or aspirin treatment. Reply:Moreover, lipid peroxidation product accumulation, MDA (Fig. 6K) and concentrations of iron (Fig. 6L) under ferroptosis conditions were also attenuated after Fer-1 or aspirin treatment. Reviewer #2: The author answered most of the questions, but the author didn't address the 4-HNE level in high glucose incubated HK2 cells. Thank you for your kind suggestion. The products of lipid peroxidation involve reactive aldehydes (e.g. 4-hydroxynonenal (4HNE) and malondialdehyde (MDA)), which increase during ferroptosis . In this study, we have added experiments on 4-HNE levels of the cells.The products of lipid peroxidation, 4-HNE(Fig. 1J) was dramatically elevated in in HG group cells.Normally, untreated control cells contained only a minimal amount of 4-HNE. The cells treated with high glucose showed the maximal level compared to NC group cells. Interestingly, aspirin treatment markedly attenuated the level compared to that in HG group cells (Fig. 4J). Moreover,the higher levels of 4-HNE were also observed in high glucose-treated HK-2 cells, and this decrease was ameliorated in high glucose-cultured cells transfected with COX2-siRNA (Fig. 5J). The lipid peroxidation product accumulation, 4-HNE(Fig.6L) under ferroptosis induced by cells cultured in 100 nm RSL-3 medium was also attenuated after Fer-1 or aspirin treatment.Simultaneously, pretreatment with transfection of COX2-siRNA reduced the concentrations of 4-HNE（Fig.7K）, which implied that transfection of COX2-siRNA could improve the states of lipid peroxidation induced by cells cultured in 100 nm RSL-3 medium. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0279010.r004
29 Nov 2022 Decision Letter - Aldrin V. Gomes, Editor A spirin Mediates Protection from Diabetic Kidney Disease by Inducing ferroptosis Inhibition PONE-D-21-40203R2 Dear Dr. Wu, 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, Aldrin V. Gomes, 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 #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: 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: This is an improved revision of the manuscript. However, minor typographical error I picked up was in results section 3.1:Another products of lipid peroxidation, 4-HNE(Fig. 1J) was dramatically "elevated in in" HG group cells. It should be "elevated in". Another minor but optional recommendation was the NC group abbreviation used in the mouse model during this study. I'm aware its the control group but it might be better if NC meaning is written in the full form at least once to inform the readers what that group is exactly. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0279010.r005
Formally Accepted
4 Dec 2022 Acceptance Letter - Aldrin V. Gomes, Editor PONE-D-21-40203R2 Aspirin Mediates Protection from Diabetic Kidney Disease by Inducing ferroptosis Inhibition Dear Dr. Wu: 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. Aldrin V. Gomes Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279010.r006

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