PONE-D-22-10698HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubulePLOS ONE

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Additional Editor Comments:

It would be preferable to mutagenize MRE and SP1 motifs in the endogenous gene promoter using genome editing techniques and examine expression levels of the SLC12A transcript.

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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

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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: Vpr-induced salt wasting and loss of NCC protein expression in mouse kidney was well studied here and, in parallel, Vpr-induced decrease in genomic bindings of MR to Slc12a3 gene location and that in the aldosterone-dependent transcriptional activity were also well demonstrated in renal cell-lines. However, considering the difficulty in detecting a change of MR genomic bindings in in-vivo DCT, the author’s hypothesis would have to be more supported by other data which associates these two parts of the study closely.

Main concerns:

1. Not only the genomic bindings of MR by Vpr treatment in human DCT cell-line, was the endogenous expression level of Slc12a3 also decreased ? At least, such changes in expression level of NCC protein and/or mRNA should be demonstrated.

2. As shown in Fig 3a, NCC protein level was clearly diminished in the kidney of Vpr-transgenic mice compared to that of wild-type mice. However, the intensity of signal in each of positive-cells in IHC and in-situ hybridization as shown in Fig. 3c and 5b seems strong almost equally in both of the groups of mice, leading to a doubt that the Vpr-induced change in renal NCC protein level would not be derived from the cellular expression level of Slc12a3 but the number of DCT cells in the kidney. At least, it should be shown that the number of DCT cells in each of the sections were not affected with the transgenic expression of Vpr.

3. About the supplemental Fig 3, the staining pattern of NCC seems rather cytoplasmic, which does not indicate strong expression of NCC in plasma membrane. More conservative statement would be more acceptable for readers.

Minor concern:

1. Some typos are observed, such as doubling of “PMC” in the reference section.

Reviewer #2: The work is very well done, experiments are well controlled and supporting evidence from various sources is nicely used. The work clearly supports an effect of VPR in the renal tubules, specifically upon MR and then NCC. My main critique of the manuscript is that this observed effect upon NCC may not be the entirety or even the major mechanism by which HIV/VPR mediated sodium wasting occurs.

Questions:

Why aren’t aldo and renin levels increased compared to controls?

Mice were fed the same chow, but was intake controlled or did actual intake vary among the mice (I expect so but not explicitly stated)? In a study of Gitelman’s patients, Lifton famously found that sodium intake among affected subjects (and heterozygotes) increased compared to unaffected individuals. Is it possible that the low sodium chow designed to induce increased aldo levels prevented us from seeing a further physiologic rise in renin/aldo? Does this in turn prevent the usual compensatory increases in sodium reabsorption along the nephron (ENaC specifically)?

I agree with the investigators that “Whether or not transgenic expresssion alters the

aldosterone/MR-mediated gene and/or protein expression of sodium transporters and exchangers

(e.g. Na+-K+-2Cl- cotransporter/NKCC2 and epithelial sodium channels/ENaCs) along the

nephron segment requires further investigation.” While the component of NCC in this phenomenon could be examined with a series of laborious thiazide treatments, this is likely to be of limited scientific value to the human disease studied due to differences in tubule contributions to sodium reabsorption across species. I would be sure to caveat my conclusions to indicate that possibility that NCC only contributes to this effect.

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Reviewer #1: Yes: Ueda Kohei

Reviewer #2: No

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HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule

PONE-D-22-10698R1

Dear Dr. Kopp,

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.

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Kind regards,

Hodaka Fujii, M.D., 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: All comments have been addressed

Reviewer #2: 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 #1: Yes

Reviewer #2: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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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: (No Response)

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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 #1: Yes

Reviewer #2: (No Response)

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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: The authors added as much data as possible to support their hypothesis and corrected their statement to be more acceptable for readers.

Reviewer #2: (No Response)

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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

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