PONE-D-23-18872EXPOSURE TO LOW LEVELS OF HEAVY METALS AND CHRONIC KIDNEY DISEASE IN THE US POPULATION: A CROSS SECTIONAL STUDYPLOS ONE

Dear Dr. Akinleye,

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. Please submit your revised manuscript by Sep 08 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Iman Al-Saleh

Academic Editor

PLOS ONE

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Additional Editor Comments:

The authors need to:

1. describe method validation for the analysis of metals and cotinine.

2. present descriptive statistics of analytes in a separate table, including the method detection limit for each metal.

3. discuss the selection of covariates in the model.

4. clarify why only the cadmium model was adjusted for cotinine; there are studies linking other metals with smoking. Have you done a correlation analysis between cotinine and metals?

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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 aim of this study was to assess any impact of low blood levels of trace heavy metals and development of CKD. The authors have used a large cohort (NHANES) and included all patients with a diagnosis of CKD, stratified by quartiles of blood Cd, Hg and Pb. The design is fine as a retrospective, descriptive cohort study in that logistic regression can be used to compare odds ratios for the lowest vs highest quartiles of blood heavy metal exposure. The aims and objectives were clearly stated and the outcome clear. I have some comments for the editor and authors to consider:

General comments:

1) I do not really see the relevance of modelling the blood metals as categorical and continuous variables. Surely as categorical has all the information you require?

2) Blood cotinine is surely a serious confounding factor and could affect the logistic regression – in model 3 there appears a significant association between Cd and CKD, but the assoc is hugely strengthened when adjusting for cotinine. Thus presumably this reflects smoking? If smoking AND cotinine are in same model then will have spurious results due to parallel/ co- correlation?

3) Lead accumulates in bone, Cd in kidney>liver and mercury in liver>brain. For the latter it also depends on whether the Hg is organic or inorganic. From fish, mostly organic. To what extent do blood levels reflect bio-accumulation in body tissues or simply acute exposure? ( I recognise you have discussed this as a limitation)

4) If associations are only with 3rd quartile and not 2nd or 4th does that indicate non-linear association with CKD risk or just an anomaly?

5) If all metals show no association after adjustment for all other factors, then do you suggest that the other factors are important (e.g. bioaccumulation with age is the biggest ‘risk’)

6) The final sentence of results and first line of conclusion are therefore at odds.

7) Methods, are the methods for metal determinations published elsewhere? They should be referred to or described.

8) It would be useful to further expand this statement and what you mean by it. Serum cotinine was used to adjust for blood cadmium level.

9) I am also unsure what this sentence actually means, especially the final part…However, blood lead level was only found to be associated with CKD in the 2nd quartile group after adjustment for blood levels of cadmium and mercury but there was a suggested dose-response relationship between the quartile groups prior to adjustment.

Reviewer #2: This is a nice paper. However, the most obvious concern lies in the originality of the paper.

Earlier publications using NHANES datasets have addressed the topic. Navas- Acien (2009) is referenced but not Ferraro et al. (2010) Research article Low level exposure to cadmium increases the risk of chronic kidney disease: analysis of the NHANES 1999-2006; Buser et al. (2016) Urinary and blood cadmium and lead and kidney function: NHANES 2007–2012; and Madrigal et al. (2019). Associations between blood cadmium concentration and kidney function in the U.S. population: impact of sex, diabetes and hypertension.

How does this paper compare to these studies. And thus, what additional information does this paper bring to the conversation re. association of low level Cd and CKD risk? The time parameters (1999 -2002) chosen for this subgroup is narrower than other studies (in some, inclusive of the time period); is there a reason for this study window e.g increased exposure risk during that time period?

Suggestion: consider a wider or different time period if there is no supportive justification for the current choice.

There is a very brief recognition of the limitation caused by the exclusion of hypertension - which can cause or arise as a consequence of CKD - but has also been linked to heavy metal exposure. Furthermore, diabetes, another cause of CKD, has also been linked to heavy metal exposure. A lack of recognition of those two chronic diseases as potential confounders are a significant limitation.

Suggestion: Consider including indices of the one/two diseases in at least one of the models to improve paper.

Reviewer #3: This is a report in which NHANES data was analyzed to determine if there are correlations between heavy metal exposure and CKD. The analysis seems to indicate that blood levels of Cd were associated with CKD. The major concern associated with this conclusion is that CKD itself could cause high blood levels of heavy metals or other toxicants due to lack of clearance. This concept is mentioned in the discussion, but the abstract needs to be updated to reflect this possibility.

1. Heavy metals are “toxicants” not “toxins.” A toxin is a biological substance (poison/venom) whereas a toxicant is something harmful in the environment.

2. Methods, 1st paragraph, last line: “individuals with missing weighing…” Should this be “weight?”

3. Cadmium and lead should not be capitalized within a sentence.

4. Was there a relationship between blood cotinine levels and CKD stage? Smoking is a major risk factor for CKD and thus, it may be more of factor in the development of CKD than Cd.

5. There are no page number or line numbers so it is difficult to provide feedback on specific grammatical errors. The discussion contains several errors that should be corrected. For example, “Chronic Renal Disease” should be chronic kidney disease.

6. It is not clear which groups are being compared for statistical significance in the tables. Is each quartile being compared to Q1 or is it compared to the previous quartile? This should be clarified in a legend associated with the table.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-18872R1EXPOSURE TO LOW LEVELS OF HEAVY METALS AND CHRONIC KIDNEY DISEASE IN THE US POPULATION: A CROSS SECTIONAL STUDYPLOS ONE

Dear Dr. Akinleye, 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.

Please submit your revised manuscript by Feb 10 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Iman Al-Saleh

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Reviewer #2 has proposed two minor additions outlined below. I would appreciate it if you could integrate these suggestions.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: 1. What are the possible explanations for the findings with cotinine?

2. Provide the reference for the classification of CKD. (KDIGO guidelines/KDOQI?)

Reviewer #3: The authors have addressed all of the previous concerns. This reviewer has no additional concerns .

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

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[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.

EXPOSURE TO LOW LEVELS OF HEAVY METALS AND CHRONIC KIDNEY DISEASE IN THE US POPULATION: A CROSS SECTIONAL STUDY

PONE-D-23-18872R2

Dear Dr. Akinleye,

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.

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Kind regards,

Iman Al-Saleh

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: