PONE-D-21-04749

APOL1 is not expressed in proximal tubules and is not filtered

PLOS ONE

Dear Dr. Bruggeman,

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.  At the same time I note that all three reviewers were enthusiastic about the project and the conclusions.

Reviewer 2 made several specific technical critiques about the work.  I would like the authors to pay special attention to the critiques of the various co-localization studies (a point that was echoed by Reviewer 3). Those analyses are central to the argument made in this MS. I agree that it might be helpful to show more micrographs, perhaps at different magnifications and with more glomeruli in the field. The authors should also add some text to address the issue that ApoL1 expression in this somewhat artificial transgenic mouse model looks a bit different than it does in humans. It would also be helpful to address the comment about the Santa Cruz antibody mentioned by the reviewer compared to the one that was used in this study, at least in the response to critiques if not in the revised MS itself. It is also important to be transparent about all statistical methods used.  Reviewer 3 also raises some other technical issues that I think will be quite straightforward to address, and that may simply be the way the figures were prepared.  That reviewer also makes some useful suggestions on spelling and formatting. 

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Additional Editor Comments (if provided):

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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: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: N/A

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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: Bruggeman et al studied APOL1 expression in tubular cells and its filtration in the tubular lumen.

Comments:

1. These investigators were pioneer to show expression of APOL1 protein in kidney cells including tubular cells. However, they have now clarified that tubular cells do not express APOL1.

2. Interestingly, they have also shown that it is not filtered. However, a possibility of its secretion by podocytes can not be excluded.

Reviewer #2: This current study tested if ApoL1 is synthesized and/or reabsorbed by proximal tubule cells. Expression of ApoL1 mRNA and protein by renal tissues of human and ApoL1 transgenic mice were examined using in situ hybridization, immunofluorescence and immunohistochemistry techniques with commercially available antibodies. The authors concluded that ApoL1 is mainly expressed in podocytes and endothelial cells, but not by tubular cells.

Overall, the findings in this study are informative, but not novel. The manuscript is logically well written, and the data is straightforward. However, supporting evidence seems insufficient because all data except for Fig 1 was generated from a transgenic mouse model. Also, the data cited that corresponds to the conclusions made regarding the filtration and reabsorption of ApoL1 part seems weak.

Comments:

1. Most studies relied on the results from BAC-ApoL1 Tg animals, in which the expression and localization of ApoL1 most likely is different from that in humans.

Indeed, mRNA and protein expression patterns of ApoL1 in liver of human and Tg mouse (Supp F2) seem quite different (disperse vs focal). Thus, human renal tissues from healthy and diseased individuals need to be thoroughly tested using the Santa Cruz antibody (lot E105260).

2. Immunofluorescence on human kidneys (Fig 1A) shows a single glomerulus with limited views of tubules. Please show the ApoL1 protein expression pattern from more tubular regions following co-staining with proximal tubule cell markers.

3. In Fig 4 (ISH on BAC-ApoL1 Tg mouse, G0? G1? G2?), decent amounts of ApoL1 mRNA expression were also observed in Nphs1 negative cells. To make it clear that the ApoL1 signals are definitively not from PT, duplex mRNA ISH for ApoL1 and PT markers need to be performed.

4. The authors showed that ApoA1 but not ApoL1 is colocalized with LT-lectin, a tubule marker (Fig 6). However, it is still not clear if positive staining of ApoA1 observed in tubular cells resulted from reabsorption from the filtrate or if it is expressed by tubular cells. Adequate measurements need to be done to clarify this issue.

We cannot rule out the possibility that a small amount of ApoL1 is released from damaged/apoptotic podocytes and reabsorbed by the proximal tubules from the filtrate.

5. If possible, additional examination of ApoL1 protein expression in human and mouse urine samples (G0, G1, and G2 genotype) would be informative.

6. Also, in Supp F1, it would be informative if the authors show the ApoL1 staining pattern in ApoL1 Tg and normal mouse renal tissues using the Santa Cruz antibody (lot E105260).

7. The statistical method used in Fig 3 has not been described.

8. The conclusions of the paper, as stated in the title is overly quite bold. The authors fall short in providing definitive evidence for the statement that proximal tubule cells neither produce nor reabsorb ApoL1 and should therefore tone down their conclusions.

Reviewer #3: Bruggeman and colleagues report that APOL1 is not expressed in proximal tubules and is not filtered. The investigators have studied (Merck) BAC/APOL1 mice, representing each of the three genotypes, crossed to FVB/N mice for 10 generations. Their prior in situ hybridization work suggests that RNA expression is confined to podocytes and endothelial cells in glomerular and peritubular capillaries, and other vessels (reference 10).

Here, the authors have done a careful analysis of various APOL1 antibodies, particularly of the Sigma polyclonal antibody. The quality of the immunostaining and in situ hybridization images is excellent. The authors conclude that bona fide APOL1 protein expression in the BAC transgenic mice is limited to podocytes and endothelial cells. They suggest that false-positivity for APOL1 immunostaining in the proximal tubule may be due to glycosylation patterns. These finding will be of value to investigators in this field.

The paper is clear, concise and elegantly written.

1) Figure 1. To my eye, it appears that there is only partial co-localization of GLEPP1 (perhaps located in peripheral portions of glomerular capillary loops) and APOL1 (possibly in the cell bodies). I wonder whether the authors agree. I agree that both proteins are in podocytes as well as other cells. I think all this might be easier to resolve if the images were larger.

2) Figure 5, Western, is confusing, because two blots were but together, with result that order of the lanes differ and protein migration differs. A single blot would strengthen the paper.

3) After all the effort to cross the mice for 10 generations onto an FVB/N background, it would be useful to know whether the kidney phenotype become more severe (more proteinuria, more glomerulosclerosis).

Minor comments.

P4,P8, capitalize Coomassie, as it is (or rather was) a place name.

P5, capitalize Lotus, as it is a genus name

PP6-7. “APOL1 expression was not detected in proximal tubules or any other nephron segment.” This sentence appears twice. More importantly, the glomerulus is part of the nephron. Suggest “or any other tubular segment.”

P7, no need to capitalize nephrin

P8, Lotus tetragonolobus, capitalize genus, italics

Bowman’s capsule > Bowman capsule. The trend is away from the possessive, as the discoverer does not own the structure. It does sound odd to say Bowman capsule after we have been saying it differently for decades.

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Reviewer #1: Yes: Pravin C. Singhal

Reviewer #2: No

Reviewer #3: No

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Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

PONE-D-21-04749R1

Dear Dr. Bruggeman,

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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Stuart E Dryer, PhD

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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 #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: (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 #2: Yes

Reviewer #3: Yes

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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 #2: In this revised version, authors addressed my concerns and made the corrections accordingly. I have no substantive suggestions to further improve the manuscript.

Reviewer #3: The authors have addressed all the issues raised. This is an important paper. Congratulations on the fine work.

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Reviewer #2: No

Reviewer #3: Yes: Jeffrey Kopp