PONE-D-25-20067 - Revision (please see the word document "Responses to reviewers")

Response to Reviewers and Editors

Response to Editors

We thank the Editor for the careful evaluation of our manuscript. Your valuable comments have helped us to substantially improve the clarity and precision of our work.

As a general note, all page and line numbers in our responses refer to the revised and marked manuscript version titled “Revised Article with Changes Highlighted”, unless otherwise specified.

1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We carefully checked the PLOS ONE’s style requirements and adjusted several headings to sentence case:

- Page 5, line 108: Cell culture

- Page 5, line 122: RNA isolation and reverse transcription

- Page 10, line 213: Quantitative real-time PCR

Additionally, we deleted the numbers from the subheadings of the Method section and we adjusted the naming of our Supporting Information files at the end of the revised manuscript in the section titled “Supporting Information” (page 42, line 955-975).

2. Thank you for stating the following in the Competing Interests section:

“I have read the journal's policy and the authors of this manuscript have the following competing interests: Oliver Domenig and Marko Poglitsch had a paid employment at Attoquant Diagnostics GmbH within the last 5 years.”

We note that one or more of the authors are employed by a commercial company: Attoquant Diagnostics GmbH

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Response: As requested, our amended Funding Statement now reads:

“Oliver Domenig and Marko Poglitsch had a paid employment at Attoquant Diagnostics GmbH within the last 5 years. Attoquant Diagnostics GmbH provided support in the form of salaries for authors [OD, MP], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the Author Contributions section.”

We reviewed our statements relating to the author contributions, and indicated the roles that these authors had in our study correctly. Thus, an update in the Author Contributions section of the online submission form is not necessary.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Response: As requested, our updated Competing Interests Statement now reads:

“Oliver Domenig and Marko Poglitsch had a paid employment at Attoquant Diagnostics GmbH within the last 5 years. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.”

We included both the updated Funding Statement and Competing Interests Statement in our cover letter.

3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

Response: We acknowledge this important note by the Editors and decided to include our entire data within the Supporting Information. Excel-file S1_Data contains primary LC-MS/MS data and S2_Data Excel-file calculation of delta delta Ct values for gene expression analyses of ACE2 and PRCP.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: Thank you for this comment. We added the information in the Supporting Information files as S5_Fig and S6_Fig. We updated the corresponding parts in the revised manuscript and deleted the phrase “data not shown”.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Response: We carefully checked the requirements for uncropped and unadjusted images underlaying blot and gel data as reported in the author guidelines. We have added the newly acquired western blot data to our raw data file. Raw images were annotated and compiled according to the author guidelines. Briefly, original images were annotated using GIMP and exported as tif-files. Tif-files were compiled into one pdf file using Adobe Acrobat. The blot/gel image data are in the Supporting Information “S1_raw_data” file. We added this information in our cover letter.

6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Response: Reviewers did not recommend to cite specific previously published works.

Response to Reviewer 1 Comments

Comment 1.1

In this manuscript, authors investigated the conversion of Ang II to Ang-(1-7) in human podocytes (hPCs) both intracellularly and at the cell surface under fluid flow shear stress (FFSS). To do this, recombinant Ang II was added in hPC lysates (representing the intracellular state) or in the culture (supernatant) of the intact cells (representing the cell surface state). The converted products Ang-(1-7) were measured by means of LC-MS/MC. RT-qPCR and immunofluorescence were used to ascertain the presence of ACE2 and PRCP in hPCs. Authors concluded that more Ang-(1-7) was formed intracellularly under the FFSS treatment, but the conversion of Ang II to Ang-(1-7) stayed steady at the cell surface regardless of FFSS. With the aid of selected enzyme inhibitors, PRCP was found responsible for the Ang-(1-7) formation inside the cells while the conversion at the cell surface involved both ACE2 and PRCP enzymes. Authors also suggested that PRCP-derived Ang-(1-7) formation was enhanced upon FFSS in the supernatants of hPCs.

The rationale of using cell lysates to represent the intracellular state or the supernatant to represent the cell surface state was not explained. If cell lysates contain all intracellular proteins and cell surface proteins, and if added Ang II and the enzyme inhibitors were internalized into the intact hPCs during the one-hour incubation time, how would the result be interpreted? Where would be the major site of the conversion, inside the cells or on the cell surface?

Response:

We thank the Reviewer for the careful evaluation of our manuscript. Your valuable comments have helped us to substantially improve the clarity and precision of our work.

As a general note, all page and line numbers in our responses refer to the revised and marked manuscript version titled “Revised Article with Changes Highlighted”, unless otherwise specified.

In addition, all raw data are now provided in the revised Supplementary Material.

Since whole-cell lysates contain both intracellular and membrane-associated proteins, we agree that the term “intracellular” was inaccurate in this context. We have carefully revised the manuscript and removed the term “intracellular” from page 9, line 223; page 13, line 281; page 15, lines 339 and 347; and page 26, line 386 of the original version. To improve accuracy, we now explicitly state “in cell lysates” where appropriate. For example, on page 16, lines 379–381, the text now reads:

“In contrast to elevated Ang-(1-7) formation in cell lysates, no effect was observed on Ang-(1-7) formation rate at the cell surface of hPC (Fig. 3B).”

Similarly, the revised text on page 19, lines 447–448, reads:

“However, Ang-(1-7) formation in hPC whole-cell lysates is mainly driven by PRCP.”

In the Discussion section (page 22, lines 502–503), we revised the sentence to:

“Our results revealed an increased conversion of Ang II to Ang-(1-7) in hPC lysates exposed to FFSS.”

For the measurements of Ang-(1–7) formation in whole-cell lysates, cells were sonicated before the addition of spiked Ang II and enzyme inhibitors. This procedure was described in the original Methods section 7.1 (page 10, lines 220–224):

“Cell suspensions were sonicated for complete dissolution using Vibra-Cell™ (Sonics & Materials Inc., Newtown, CT, USA) and normalized to total protein concentration determined using the BIO-RAD Protein Assay Dye with bovine serum albumin as protein standard (cat. no. 5000006, Hercules, CA, USA).”

Therefore, internalization of Ang II is not relevant in this context.

In contrast, for extracellular Ang-(1–7) formation in supernatants, Ang II and enzyme inhibitors were added to intact cells. During the one-hour incubation period, receptor-mediated internalization of Ang II could have occurred. However, such potential internalization would have affected both experimental conditions (control and FFSS) equally. Importantly, Ang II was added in saturating concentrations, and residual substrate levels were measured after incubation to confirm that enzyme activities were not substrate-limited. In addition, there is, to our knowledge, no rationale for enzyme inhibitors to undergo internalization in intact cells.

Regarding the Reviewer’s question about the major site of Ang II conversion to Ang-(1-7), we consider it most likely to occur inside the cells. This is supported by the low contribution of ACE2 to Ang-(1-7) formation in whole-cell lysates and the presumably higher intracellular content of PRCP. A direct quantitative comparison between lysates and supernatants is not feasible, however, since Ang-(1-7) formation in lysates was normalized to protein content, whereas absolute values were determined in supernatants.

Comment 1.2

The presentation and interpretation of the data were somewhat convoluted and hard to follow.

Response: We thank the reviewer for this overall comment. We acknowledge, that the flow of the manuscript may have been difficult to follow for the reader. To improve clarity and readability, we thoroughly revised the manuscript and expanded on the main findings and their implications within the Results section (see also Reviewer 2, comment 6).

To enhance data presentation and to facilitate interpretation, we have incorporated Supplementary Figure S2A-D into the main text of the revised manuscript, now presented as Figure 2, in line with the recommendation of Reviewer 2.

In addition, we restructured the manuscript to provide a more logical sequence of methods and results. Specifically, the methodology and results of the inhibitor testing were moved forward to directly follow enzyme characterization. This section now appears as Methods section 4 (“Inhibition of Ang-(1-7) formation by different inhibitors,” page 7, line 163 ff.) and in the revised Results section as “Enzymes involved in Ang-(1-7) formation from substrate Ang II” (page 15, line 339 ff.).

Finally, the presentation of the data in the original Figures 4 and 5 (now revised Figures 5 and 6) has been improved for greater clarity. For further details on these revisions, please see our response to Comment 1.12.

Comment 1.3

Authors reported that the PRCP mRNA expression in hPCs was slightly decreased upon FFSS, yet the conversion of Ang II to Ang-(1-7) was increased intracellularly (cell lysate) under FFSS. This conversion was mainly driven by PRCP in cell lysates and enhanced at the cell surface (in supernatant) under FFSS. Please comment.

Response:

We thank the Reviewer for this insightful comment. In our measurements of PRCP mRNA expression in cell lysates, we observed a slight decrease under FFSS (revised Figure 4). In contrast, the total Ang-(1–7) formation rate was significantly increased under FFSS conditions (revised Figure 3). As the Reviewer correctly notes, Ang-(

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PONE-D-25-20067R1 – Revision

Response to Reviewers and Editors

Response to Editors

We thank the Editor for the thorough evaluation of our manuscript. In the following, we address the general remarks provided:

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Response: Thank you for the advice. However, the Reviewers did not propose any specific references.

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.

Response: We carefully reviewed our reference list and checked for retracted articles. We did not find any retracted article in our list. However, reference number 65 (Velez JC, Bland AM, Arthur JM, Raymond JR, Janech MG. Characterization of renin-angiotensin system enzyme activities in cultured mouse podocytes. Am J Physiol Renal Physiol. 2007;293(1): F398–407. Epub 20070411. doi: 10.1152/ajprenal.00050.2007. PubMed PMID: 17429035.) was corrected in 2008, as the authors had mistakenly labeled a measured peptide as Ang-(1-9), which was later found to be Ang-(2-10) [1]. The correction does not affect our citation of the study. Therefore, we have retained this reference.

We also identified an updated PMID number for reference number 2 and updated it in our reference list (Inagami T. The renin-angiotensin system. Essays Biochem. 1994; 28:147–64. PubMed PMID: 7925317).

Response to Review Comments to the Author

Response to Reviewer #2: (No Response)

Response: We are glad that we were able to successfully address all of Reviewer’s #2 questions and comments.

Response to Reviewer #3:

Due to limitations in study design some comments could not be responded successfully.

Response: We thank the Reviewer #3 for carefully reading our manuscript and evaluating our responses to the Reviewers #1 and #2. Your comments helped us further improve our manuscript.

As a general note, all page and line numbers in our responses refer to the latest revised and marked manuscript version titled “Revised Article with Changes Highlighted”, unless otherwise specified.

Comment 1.1

This comment has been partially addressed.

Response: We thank the author for this comment and noticed that we need to explain the methodology in more detail.

a. What was the composition of the buffer used for the preparation? did it have any detergents? ionic strength?

Response: We used PBS (pH 7.4) to assess Ang-(1-7) formation in hPC cell lysates. Cells were scraped from the slides and transferred to PBS prior to sonification as described in the original manuscript on page 9 lines 220-221:” Cells were scratched from slides and transferred to a vial containing PBS.” We added the product information when first mentioned in the manuscript Method section – immunofluorescence in lines 133-134, where it now reads: “Medium was discarded and cells washed twice with phosphate buffered saline (PBS, cat. no. 14190094, Thermo Fisher Scientific, Waltham, MA, US).”

No detergents were used in the sample preparation procedure of cell lysates. To calculate the ionic strength, we relied on the manufacturer's specifications for the salt composition of PBS, assumed an ideal diluted solution and that all phosphorous compounds are dissociated completely. The resulting ionic strength for PBS at pH 7.4 is 166 mM.

b. Cells suspensions were sonicated for complete dissolution... I think complete dissolution of membranes is not possible with this methodology.

Response: We thank the reviewer for this comment. Cells were lysed using Vibra-Cell™ and membranes were destroyed by sonication using this methodology. We agree with the Reviewer, that the word “complete” cannot be used with absolute certainty in this context and therefore, deleted it from the sentence. The revised manuscript now reads in lines 221-223: “Cell suspensions were sonicated for dissolution using Vibra-Cell™ (Sonics & Materials Inc., Newtown, CT; US) and centrifugated at 16260 rcf for 10 min at 4°C.”

c. Was there any centrifugation step involved?

What was the duration and conditions of centrifugation? This is important to know what type of membranes author worked with. If no centrifugation was used the protocol seems not appropriate.

Response: That is a valuable comment. Indeed, we performed a centrifugation step. We sincerely apologize for our inaccuracy in the description of the methodology. For the cell lysates sample preparation, samples were centrifuged at 4°C for 10 minutes and 16260 rcf after sonication. Supernatants were collected and protein concentration quantified. We have amended the methodology in the revised manuscript. It now reads in lines 221-226: “Cell suspensions were sonicated for dissolution using Vibra-Cell™ (Sonics & Materials Inc., Newtown, CT; US) and centrifugated at 16260 rcf for 10 min at 4°C. Supernatants were collected and normalized to total protein concentration determined using BIO-RAD Protein Assay Dye with bovine serum albumin as protein standard (cat. no. 5000006, Hercules, CA, US).”

Comment 1.3

This comment has not been addressed. Authors postulate now the participation of another enzyme that may contribute to the increased Ang-(1-7) formation observed under FFSS in cell lysates. Which enzyme do authors considered?

Response: We recognize that further consideration and clarification are needed regarding additional potential enzymes involved in Ang-(1-7) formation, particularly as Reviewer #4 has raised a similar concern. We agree that at least a literature-based hypothesis, as suggested by Reviewer #4, is necessary to address this mechanistic gap. Thus, we conducted an extensive literature search to identify enzymes that could potentially account for the observed conversion of Ang II to Ang-(1-7) besides ACE2, PRCP and PREP:

Cathepsin A (CTSA), a lysosomal enzyme widely expressed in human tissues including the kidney [2], was initially proposed as a candidate for Ang-(1-7) formation. Early studies by Grynbaum and Marks reported that Cathepsin A from rat brain could release the C-terminal Phe from Ile^5-Ang II, producing Ang-(1-7) [3]. In contrast, Miller et al. found that Cathepsin A from porcine kidney cleaved Ang I to Ang-(1-9) and to Ang II, but did not hydrolyze Ang II [4]. Subsequent studies similarly highlighted Ang I as a primary substrate of Cathepsin A [5].

Grobe et al. reported that nephrolysin (NEP), thimet oligopeptidase (THOP1), and neurolysin (NLN) can generate Ang-(1-7), though they cleave Ang I and not Ang II [6]. Both NLN and THOP1 cleave Ang I at the Pro-Phe bond to form Ang-(1-7) [7, 8]. NLN is ubiquitously expressed, including in the kidney [9], but is primarily studied in the brain, where it is associated with stroke [10, 11]. While NLN can inactivate Ang II [8, 11, 12], this produces Ang-(1–4), not Ang-(1–7) [13]. Consequently, it is not a potential enzyme for our investigated conversion of Ang II to Ang-(1-7).

Velez et al. suggested that THOP1 may also convert Ang II to Ang-(1-7), alongside ACE2, RPCP, and PREP [14]. Early assays found no Ang II cleavage by recombinant rat THOP1 [15], while later studies suggested inefficient cleavage due to low catalytic efficiency [16]. Since existing statements are contradictory, we conclude that it remains unclear whether THOP1 actually hydrolyzes Ang II besides Ang I. Furthermore, no studies have identified specific Ang II–derived products of THOP1. Thus, THOP1 might inactivate Ang II by generating Ang-(1–4) or it might produce Ang-(1–7).

Overall, current evidence does not conclusively show that any of these enzymes convert Ang II to Ang-(1–7). Further studies are needed to define the enzymes regulating the RAS in podocytes, particularly experimentally assessing THOP1 and Cathepsin A under our conditions in hPC. We addressed these considerations in the revised Discussion section, which now reads in lines 522-533: “Additional enzymes involved in the degradation of angiotensin peptides that should be further investigated include Cathepsin A (CTSA; EC 3.4.16.5) and thimet oligopeptidase (THOP1; EC 3.4.24.15). Although CTSA is mainly known to hydrolyze Ang I [79], early studies suggest it may also cleave Ang II to Ang-(1–7) [80]. THOP1 is well established in cleaving Ang I to Ang-(1–7) [81,82], and further studies have indicated that it can also cleave Ang II, albeit inefficiently due to its low catalytic efficiency [83]. However, potential Ang II–derived cleavage products of THOP1 have not yet been identified. Future research might therefore specifically examine THOP1 and Cathepsin A under the experimental conditions in hPC to clarify their potential roles in local RAS regulation and defining more precisely the enzymatic mechanisms underlying Ang II processing in hPC.”

Comment 1.4

I concur with Reviewer 1 int that Ang II levels used were too high to make an extrapolation to in vivo settings

Response: We thank the Reviewer for raising this concern once again. As noted in our response to Comment 1.4 in our first revision, we agreed and acknowledged that the concentration of Ang II used in our experiments does not reflect in vivo peptide levels and represents a limitation of the study. To more clearly emphasize the importance of this comment, we have highlighted this limitation explicitly in the discussion section of the newly revised manuscript. It now reads in lines 604-610: “We deliberately chose a high Ang II concentration of 1µg/mL to evaluate catalytic capacities under standardized conditions avoiding artificial limitations on enzyme activity. Although the exogenously applied Ang II concentration does not reflect physiological in vivo peptide levels, this approach enabled us to use spiked Ang II as a substrate, allowing clear identification of only the spiked product via mass spectrometry and the precise calculation of enzyme activity.”

Comment 1.15

ACE2 activity coming from the cell surface cannot be evaluated with this protocol since all membranes were mix together after sonication and centrifugation of lysates

Response: Two different experimental setups were used to assess enzyme activity in cell lysates and on the cell surface. For cell lysate measurements, cells were sonicated as described in the Method section “Cell lysates sample preparation”. Enzyme activities at the cell surface were determined using intact cells as described in the Method section “Cell supernatants sample preparation”, where Ang-(1-7) formation was measured in supernatants of intact cells. To highlight the use of cell supernatants we wrote in the revised manuscript in lines 238-241: “100µL inhibitor solutions were applied each into one chamber of the cassette onto hPC and incubated for 1 h at 37 °C. Supernatants were collected and, following stabilization by acidification, Ang-(1-7) formation was quantified by LC-MS/MS (Methods 7.3).”

Response to Reviewer #4:

This study clarifies how fluid shear stress (FFSS) regulates Angiotensin-(1-7) formation in human podocytes. The authors show that the intracellular enzyme PRCP, not ACE2, is the dominant source of Ang-(1-7) production, a potentially protective pathway enhanced by FFSS. Here, a few concerns regarding data interpretation and discussion need to be addressed.

Response: We appreciate the Reviewer’s overall feedback, as well as his thorough and thoughtful comments provided below. They have been extremely valuable and we hope that we have addressed them sufficiently.

As a general note, all page and line numbers in our responses refer to the latest revised and marked manuscript version titled “Revised Article with Changes Highlighted”, unless otherwise specified.

1. The residual Ang-(1-7) formation in hPC lysates after dual ACE2 and PRCP inhibition (Fig 5) is noted to be upregulated by FFSS. This finding is a key mechanistic gap, the authors hypothesize another enzyme is involved but offer no data. Experimental or literature-based discussion is essential to hypothesize which other peptidases (e.g., Cathepsin A) could account for this activity, thereby providing a complete map of the RAS balance in the podocyte and guiding future research.

Response: We thank the Reviewer for this valuable comment and acknowledge the importance of further elaborating on additional peptidases involved in Ang-(1-7) formation. We conducted an extensive literature search to identify enzymes that might account for the observed conversion of Ang II to Ang-(1-7).

Cathepsin A (CTSA), a lysosomal enzyme widely expressed in human tissues including the kidney [2], was initially proposed as a candidate for Ang-(1-7) formation. Early studies by Grynbaum and Marks reported that Cathepsin A from rat brain could release the C-terminal Phe from Ile^5-Ang II, producing Ang-(1-7) [3]. In contrast, Miller et al. found that Cathepsin A from porcine kidney cleaved Ang I to Ang-(1-9) and to Ang II, but did not hydrolyze Ang II [4]. Subsequent studies similarly highlighted Ang I as a primary substrate of Cathepsin A [5].

Grobe et al. reported that nephrolysin (NEP), thimet oligopeptidase (THOP1), and neurolysin (NLN) can generate Ang-(1-7), though they cleave Ang I and not Ang II [6]. Both NLN and THOP1 cleave Ang I at the Pro-Phe bond to form Ang-(1-7) [7, 8]. NLN is ubiquitously expressed, including in the kidney [9], but is primarily studied in the brain, where it is associated with stroke [10, 11]. While NLN can inactivate Ang II [8, 11, 12], this produces Ang-(1–4), not Ang-(1–7) [13]. Consequently, it is not a potential enzyme for our investigated conversion of Ang II to Ang-(1-7).

Velez et al. suggested that THOP1 may also convert Ang II to Ang-(1-7), alongside ACE2, RPCP, and PREP [14]. Early assays found no Ang II cleavage by recombinant rat THOP1 [15], while later studies suggested inefficient cleavage due to low catalytic efficiency [16]. Since existing statements are contradictory, we conclude that it remains unclear whether THOP1 actually hydrolyzes Ang II besides Ang I. Furthermore, no studies have identified specific Ang II–derived products of THOP1. Thus, THOP1 might inactivate Ang II by generating Ang-(1–4) or it might produce Ang-(1–7).

Overall, current evidence does not conclusively show that any of these enzymes convert Ang II to Ang-(1–7). Further studies are needed to define the enzymes regulating the RAS in podocytes, particularly experimentally assessing THOP1 and Cathepsin A under our conditions in hPC. We addressed these considerations in the revised Discussion section, which now in lines 522-533: “Additional enzymes involved in the degradation of angiotensin peptides that should be further investigated include Cathepsin A (CTSA; EC 3.4.16.5) and thimet oligopeptidase (THOP1; EC 3.4.24.15). Although CTSA is mainly known to hydrolyze Ang I [79], early studies suggest it may also cleave Ang II to Ang-(1–7) [80]. THOP1 is well established in cleaving Ang I to Ang-(1–7) [81,82], and further studies have indicated that it can also cleave Ang II, albeit inefficiently due to its low catalytic efficiency [83]. However, potential Ang II–derived cleavage products of THOP1 have not yet been identified. Future research might therefore specifically examine THOP1 and Cathepsin A under the experimental conditions in hPC to clarify their potential roles in local RAS regulation and defining more precisely the enzymatic mechanisms underlying Ang II processing in hPC.”

2. This study's conclusion rests on the idea that the PRCP-driven Ang-(1-7) increase is a "renoprotective" shift in the RAS balance, but this is never demonstrated functionally in the model. Determine if PRCP inhibition significantly exacerbates this FFSS-induced inj

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