Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis

James F. Dylewski; Pantipa Tonsawan; Gabriela Garcia; Linda Lewis; Judith Blaine

doi:10.1371/journal.pone.0230401

Peer Review History

Original SubmissionFebruary 24, 2020
6 Mar 2020 Decision Letter - Zhanjun Jia, Editor PONE-D-20-05364 Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of immune-mediated kidney disease PLOS ONE Dear Dr Dylewski, 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. We would appreciate receiving your revised manuscript by Apr 20 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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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: Yes Reviewer #2: Partly ******** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is an interesting and significant research work. The authors demonstrated that induction of anti-GBM nephritis is not dependent on podocyte FcRn and lack of podocyte FcRn was protective in immune mediated kidney disease that is dependent on an autologous phase. Their findings suggested that specific therapies should be tailored according to the specific disease and the certain stage. The experiments were performed by utilizing two different mouse models of immune-mediated nephritis, the methodologies used were adequate. Suggestions: 1. Since authors mentioned in the discussion that podocytes are involved in making glomerular crescents and FcRn deficiency alters mobility and actin dynamics in cultured podocytes, it seems logical to check the situation of podocyte injury in vivo in their own FcRn KO mice with or without anti-GBM antibody treatment. 2. The NTS model also has a heterologous phase although within a few days, since authors propose podocyte FcRn as a renoprotective factor mainly involved in autologous phase but not the heterologous phase, it is better to examine the effects of podocyte-specific knockout of FcRn in the heterologous phase of NTS model. 3. The Fig 4 lacks the panel H. 4. Please explain why the control groups were missed in the in vivo experiments (anti-GBM nephritis and NTS neohritis models). 5. At the beginning of this muscript, authors mentioned that immune-mediated nephritis is the third leading cause of end stage kidney disease in the United States (1). While in the Ref 1, authors seems not mention the third leading cause of end stage kidney disease in US. Could the authors provide the detail of its source? Reviewer #2: In the manuscript entitled as “Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of immune-mediated kidney disease”, the authors first proved that podocyte could function as antigen presenting cells. FcRn was implicated in this process by using FcRn KO podocytes. Results in the NTS mice model demonstrated that podocyte-specific knockout of FcRn reduced albuminuria, glomerulosclerosis, and crescent formation. They fairly interpreted the data and clearly presented the results in the manuscript. The quality of the manuscript could be further improved once the following issues had been addressed. Major concerns: 1. The quality of the images should be improved. 2. Please provide profile of the KO podocyte, e.g. the knockout efficiency, the cellular characteristics of the podocytes, in Figure 1. Does the FcRn knockout affect the morphology of the podocytes? 3. Please prove that FcRn was specifically knocked out in the podocyte in the animal model. 4. The authors collected urine for the analysis of albuminuria. How was the urine collected? Was it 12h or 24h urine? 5. Other than what described in Figure 3, is there any abnormality in the renal function in the FcRn fl/fl;Pod-Cre mice? 6. Normal controls that were not injected with anti-GBM antibody should be included in Figure 3 &4 to prove the establishment of model. 7. The quality of the immunofluorescent staining shown in Figure 3D should be improved, as the green fluorescence with the similar intensity as the positive staining also present in the tubular. Minor concerns: 1. Was the person who performed the histological scoring of glomeruli blinded to the grouping? 2. Please describe the successful rate and mortality rate of anti-GBM nephritis model and nephrotoxic serum nephritis models in the Methods section. How many mice per group were used at the start of modeling? 3. In the Methods for the immunofluorescence for fixed cells, please name the type of normal serum used for blocking. 4. In Figure 1, the authors found that IFNγ-stimulation induced significant increase of MHCII in the podocytes. Please specify the control used for comparison. 5. Please discuss the results in Figure 1. 6. The numbering of the images in Figure 3 was not consistent with the figure legend. 7. What is the unit of the Y axis in Figure 3B? 8. Please counter-stain the nuclei in the Figure 3C. ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0230401.r001
Revision 1
3 Dec 2020 Author Response We appreciate the reviewers’ detailed and thoughtful comments that have helped to improve this manuscript. Below is our response to the reviewers: Reviewer #1: Since authors mentioned in the discussion that podocytes are involved in making glomerular crescents and FcRn deficiency alters mobility and actin dynamics in cultured podocytes, it seems logical to check the situation of podocyte injury in vivo in their own FcRn KO mice with or without anti-GBM antibody treatment. We have provided histologic analysis of control and podocyte specific FcRn KO mice injected with saline as well as a non-specific rabbit IgG control in supplemental figure 2. We observed no difference in glomerulosclerosis scores between the control and podocyte specific FcRn KO mice injected with saline or rabbit IgG. The NTS model also has a heterologous phase although within a few days, since authors propose podocyte FcRn as a renoprotective factor mainly involved in autologous phase but not the heterologous phase, it is better to examine the effects of podocyte-specific knockout of FcRn in the heterologous phase of NTS model. Our aim in this paper was to determine the ability of podocytes to act as antigen presenting cells and whether targeting the podocytes ability to present antigen could attenuate the adaptive immune response leading to glomerulonephritis. The heterologous phase of the NTS consists of the innate immune response which should not be dependent on antigen presentation by podocytes. As such, we feel the study of the podocyte FcRn on the innate immune response extends beyond the scope of this paper and should be investigated separately. The Fig 4 lacks the panel H. This was a labeling error and it has been corrected. Please explain why the control groups were missed in the in vivo experiments (anti-GBM nephritis and NTS nephritis models). The in vivo experiments were performed in podocyte specific FcRn knockout mice and control mice (FcRn fl/fl;+/+) which lacked the cre expression and thus had normal FcRn expression. All analysis compared the control mice (FcRn fl/fl;+/+) and the podocyte specific FcRn knockout mice (podoFcRn fl/fl;Cre/+) to determine the relative effect podocyte FcRn had on disease severity. “Sham” controls of intravenous saline and non-specific IgG were performed and now included in Supplemental Figure 2. At the beginning of this manuscript, authors mentioned that immune-mediated nephritis is the third leading cause of end stage kidney disease in the United States (1). While in the Ref 1, authors seems not mention the third leading cause of end stage kidney disease in US. Could the authors provide the detail of its source? The United States Renal Data Systems (USRDS), lists glomerulonephritis as the third leading cause for End Stage Kidney disease in the United States. We have changed our wording of “immune mediated kidney disease” to “glomerulonephritis” in order to be more specific to the type of diseases we are investigating. Reviewer #2 Major concerns: The quality of the images should be improved. Representative images using the highest resolution of the images available were included in this resubmission. Furthermore, the PDFs containing these images are now saved as ‘Press Quality’ to ensure the resolution is preserved during the uploading process. Some images are more pixelated because these images were obtained at a lower power magnification that resulted in the loss of resolution when zoomed in to focus on individual glomeruli. Please provide profile of the KO podocyte, e.g. the knockout efficiency, the cellular characteristics of the podocytes, in Figure 1. Does the FcRn knockout affect the morphology of the podocytes? WT and KO podocytes were characterized in the following paper: Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo Dylewski et al., PLoS One 2019, PMCID: PMC6392300. KO podocytes have similar morphology to KO podocytes but do have differences in the actin cytoskeleton (Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton. Tonsawan et al., AJP Cell Physiol, 2019 PMCID: PMC6879880). Please prove that FcRn was specifically knocked out in the podocyte in the animal model. Podocyte-specific KO mice were characterized in: Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo Dylewski et al., PLoS One 2019, PMCID: PMC6392300. Additional characterization as well as details on genotyping and generation of these mice are described in Generating a Podocyte-Specific Neonatal Fc Receptor (FcRn) Knockout Mouse. Blaine J. Methods Molecular Biology, Vol. 2224, ShreeRam Singh et al. (Eds): Mouse Genetics, Springer Nature, 2020, accepted. In this chapter we show that staining for FcRn in vivo is absent in the glomerular locations in which podocytes are found in podocyte-specfic FcRn KO mice but is present in the endothelium. We also demonstrate that staining for FcRn is minimal in podocytes isolated from podocyte-specific FcRn KO mice compared to podocytes isolated from control mice. The authors collected urine for the analysis of albuminuria. How was the urine collected? Was it 12h or 24h urine? The urine was a spot (random collection) Other than what described in Figure 3, is there any abnormality in the renal function in the FcRn fl/fl;Pod-Cre mice? In our previous paper, Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo Dylewski et al., PLoS One 2019, PMCID: PMC6392300, we show that 3 month old podocyte-specific FcRn KO mice have normal levels of serum albumin, similar albuminuria and similar GFRs compared to control mice. In the experiments described in this paper we used mice that were 10 – 12 weeks of age. Normal controls that were not injected with anti-GBM antibody should be included in Figure 3 &4 to prove the establishment of model. The in vivo experiments were performed in podocyte specific FcRn knockout mice and control mice (FcRn fl/fl;+/+) which lacked the cre expression and thus had normal FcRn expression. All analysis compared the control mice (FcRn fl/fl;+/+) and the podocyte specific FcRn knockout mice (podoFcRn fl/fl;Cre/+) to determine the relative effect podocyte FcRn had on disease severity. “Sham” controls of intravenous saline and non-specific IgG were performed and now included in Supplemental Figure 2. The quality of the immunofluorescent staining shown in Figure 3D should be improved, as the green fluorescence with the similar intensity as the positive staining also present in the tubular. The staining in Figure 3D has been redone. Minor concerns: 1. Was the person who performed the histological scoring of glomeruli blinded to the grouping? Yes, all histologic analysis was performed in a blinded fashion. 2. Please describe the successful rate and mortality rate of anti-GBM nephritis model and nephrotoxic serum nephritis models in the Methods section. How many mice per group were used at the start of modeling? For the anti-GBM mice mortality was as follows: one control and two podocyte-specific FcRn Ko mice died during the experiment. Starting numbers were 11 control and 12 podocyte-specific FcRn KO mice. For the NTS experiments, one control and one podocyte-specific FcRn KO mouse died so starting numbers were as follows: 9 control and 8 podocyte-specific FcRn KO mice. This information has been added to the Methods section. 3. In the Methods for the immunofluorescence for fixed cells, please name the type of normal serum used for blocking. This section was included in error. In this manuscript there is no immunofluorescence on fixed cells so this section of the Methods has been removed. 4. In Figure 1, the authors found that IFNγ-stimulation induced significant increase of MHCII in the podocytes. Please specify the control used for comparison. The control used was podocytes treated with regular media without any IFN�. 5. Please discuss the results in Figure 1. It is established that cells, including podocytes, can express MHCII after exposure to INF�. Since FcRn is involved in antigen loading on MHCII, the experiments in Figure 1 were performed to ensure that knockout of FcRn did not alter the podocytes ability to express MHCII and co-stimulatory markers and thus impair the FcRn KO podocytes ability to present antigen. This has been added to the discussion section. 6. The numbering of the images in Figure 3 was not consistent with the figure legend. The numbering has been changed to ensure they are correct and aligned with what is described in the text. 7. What is the unit of the Y axis in Figure 3B? The units for the Y axis in Figure 3B is OD450 and have been added to the figure. 8. Please counter-stain the nuclei in the Figure 3C. Figure 3C is demonstrating the presence of C3 within the kidney. Since C3 deposition is not specific to the location of cells, nuclei staining was not performed in this portion of the analysis. Staining for actin was performed to identify structural components within the kidney. https://doi.org/10.1371/journal.pone.0230401.r002
9 Dec 2020 Decision Letter - Zhanjun Jia, Editor Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis PONE-D-20-05364R1 Dear Dr. Dylewski, 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, Zhanjun Jia 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 ******** 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) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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) ****** 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) ****** 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: No https://doi.org/10.1371/journal.pone.0230401.r003
Formally Accepted
14 Dec 2020 Acceptance Letter - Zhanjun Jia, Editor PONE-D-20-05364R1 Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis. Dear Dr. Dylewski: 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. Zhanjun Jia Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230401.r004

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