We would like to thank the Editor and the Reviewers for their positive and encouraging evaluation of our manuscript. We appreciate the insightful and constructive comments provided, which have significantly helped us improve the quality and clarity of our work.

In response to the reviewers’ suggestions, we have performed additional experiments, including podocin and HA immunostaining, re-evaluated our statistical analyses, and provided raw data for transparency. We have also added a new supplementary figure illustrating our methodology and included additional references. The manuscript has been extensively edited for clarity, flow, and grammatical consistency.

Our point-by-point responses to the specific concerns raised by the reviewers are detailed below.

Response to Reviewer 1

1. Quantification of imaging data and sample sizes:

Many key observations—particularly loss of podocin or WT1 staining—are presented solely as representative images. Quantitative morphometric data and reporting of sample sizes are necessary.

Response: We appreciate the reviewer’s constructive suggestion. To enhance the robustness of our findings, we have now performed a quantitative analysis of podocin signal intensity for Figures 2, 5, 6, S2, S3, and S4.

To ensure accurate quantification, we addressed two technical challenges: 1) podocin intensity varies with the area of podocyte clusters within a given section, and 2) the baseline differentiation of organoids can vary between batches. To correct for these factors, we normalized the podocin-positive intensity to the HA-positive area in adjacent sections. The HA-tag, fused to a mitochondrial protein specifically in podocytes, serves as a stable internal marker because its expression is more resistant to LMB2-induced injury compared to podocyte differentiation markers. For S3 Figure, podocin intensity was normalized to the total organoid area, as NEP25 organoids lack the HA tag.

The quantitative data for each individual organoid are detailed in S2 Table, which allows for a clear assessment of the sample size for each experimental group. Typically, we analyzed n = 2-4 organoids per condition to ensure reproducibility. While the sample size for some groups was constrained by the complexity of the organoid assays, the quantitative results consistently demonstrate that podocin expression is significantly suppressed by LMB2 in both NEP25/RiboTag and chimeric organoids. Furthermore, this suppression was not attenuated by CBX or other tested inhibitors, supporting our qualitative observations. We have updated the Methods section and Figure Legends to reflect these quantitative analyses.

2. Data availability requirements:

Raw data (e.g., Ct values for qPCR, numerical replicate values, or quantifications of immunostaining) should be provided.

Response: In compliance with PLOS ONE’s data policy, we have provided all raw data. S3 Table now includes Ct values for all qPCR experiments, and S2 Table contains numerical replicate values and quantifications for all podocin immunostaining.

3. Statistical analysis considerations:

Assumptions (e.g., normality) for polysome qRT-PCR experiments are not discussed.

Response: We thank the reviewer for this important point. Since the original qPCR data exhibited a right-skewed distribution, we performed logarithmic transformations to stabilize the variance and approximate a normal distribution.

In the revised Fig. 3, we now present scatter plots with geometric means. While log-transformation was generally effective, we evaluated the normality of residuals and noted that for certain genes (e.g., Nphs1 and Wt1 in Fig. 3d), the residuals remained slightly skewed. However, alternative transformations (square-root or reciprocal) provided a marginally better fit to the normal distribution. However, the statistical significance remained unchanged regardless of the transformation method used. For instance, the adjusted P-values for Nphs1, Nphs2, Wt1, and Gadd45b remained consistently below 0.05. This confirms the robustness of our conclusions. We have added a statement regarding these transformations to the Figure 3 legend.

4. Clarity and style of writing:

Several sentences are overly long or densely structured. Minor grammatical and stylistic inconsistencies are present.

Response: We have conducted a comprehensive revision of the manuscript to improve readability. Long sentences have been shortened, and we have refined technical descriptions to be more concise. All Figure Legends have also been revised for clarity regarding experimental groups and statistical analyses.

5. Mechanistic implications:

The Discussion could benefit from a more explicit statement that the current data rule out some pathways only partially.

Response: We agree with the reviewer’s insightful point. While our inhibitor experiments (including gap junction blockade) did not show a rescue effect, we acknowledge that this does not constitute an exhaustive mechanistic screen. We have added a statement to the Discussion clarifying that these pathways are only partially ruled out and acknowledging this as a limitation of the study.

Response to Reviewer 2

1. References in Introduction:

The corresponding references should be inserted after the last sentence for the first and second paragraphs.

Response: We have now included the relevant references at the end of the first and second paragraphs of the Introduction as requested.

2. Methods details and graphical summary:

Provide more details about IP and RNeasy Micro Kit. Can the authors present a graphical summary of the methods?

Response: We have expanded the Methods section to include more detail on the immunoprecipitation procedure and the use of the RNeasy Micro Kit, including additional citations. Furthermore, we have added S7 Figure, which provides a schematic overview of the polysome analysis in chimeric organoids to facilitate reader understanding.

3. Results - Statistical identification:

Please identify the statistically different groups in the figures.

Response: We have updated Figure 3 to clearly indicate statistically significant groups using asterisks (*).

We believe that these revisions have strengthened the manuscript and addressed all the concerns raised by the reviewers. We hope the revised version is now suitable for publication in PLOS ONE.

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May 28, 2026

Dear Editor and Reviewers,

We would like to express our sincere gratitude to the reviewers for their constructive comments and highly positive evaluation of our manuscript entitled "Podocyte injury damages podocytes in chimeric organoids" (Manuscript ID: PONE-D-25-60672R).

We have carefully addressed all the points raised by Reviewer #3. The manuscript has been revised accordingly, and we believe these changes have significantly improved the clarity and quality of our manuscript.

Response to Academic Editor

Journal Requirement / Recommendation: 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.

Response: We sincerely appreciate the editor’s valuable recommendation to utilize protocols.io to enhance reproducibility. Regarding the protocol for generating the kidney organoids, which is a key characteristic of our study, we would like to clarify that the detailed step-by-step procedures have already been fully established and previously published by our co-authors (referred to as Ref. 18 in the manuscript). Because this robust protocol is already available to the public and properly cited in our Methods section, we have not deposited it as a new protocol on protocols.io. We hope the editor finds this explanation acceptable, as the reproducibility of the method is already well-secured through our previous publication.

Below is our point-by-point response to the reviewer’s comments.

Response to Reviewer #3

General Comment:

The manuscript presents a well-designed experimental study investigating the propagation of podocyte injury using chimeric kidney organoids. The use of a mosaic organoid system to isolate direct cell–cell interactions is a notable strength, and the findings provide meaningful insight into mechanisms underlying local injury spread in kidney disease. The combination of imaging, molecular analysis, and functional assays supports the conclusions. Overall, the study is of good quality, and only minor revisions are required to further improve clarity and presentation.

Response: We sincerely thank the reviewer for the highly encouraging feedback and appreciation of our mosaic organoid system. We agree that the suggested minor revisions greatly enhance the clarity and presentation of our findings.

Comment 1: Some sentences, particularly in the introduction and discussion sections, are slightly long and could be simplified to improve readability.

Response: We agree with the reviewer's assessment. We have simplified several long and complex sentences throughout the Introduction and Discussion sections to enhance readability.

• In the Introduction [Lines 50-52]: We split the sentence regarding LMB2 clearance into two separate sentences:

o Old: "Notably, a single injection of LMB2 results in progressive kidney injury over several weeks, even though the toxin is rapidly cleared and podocytes with activated caspase are eliminated within seven days."

o Revised: "Notably, a single injection of LMB2 results in progressive kidney injury extending over several weeks. This progression occurs despite the rapid clearance of the toxin and the elimination of caspase-activated podocytes within seven days."

• In the Discussion [Lines 280-282]: We similarly simplified the sentence describing injury propagation:

o Old: "Our findings indicate that podocytes directly injured by LMB2 can induce secondary damage to neighboring, initially intact podocytes within chimeric organoids, despite the absence of glomerular filtration or other glomerular cell types."

o Revised: "Our findings indicate that podocytes directly injured by LMB2 induce indirect damage to neighboring, initially intact podocytes within chimeric organoids. Importantly, this propagation occurs even in the absence of glomerular filtration or other glomerular cell types."

Comment 2: The distinction between “indirect injury,” “secondary injury,” and “bystander effect” could be clarified and used more consistently throughout the manuscript.

Response: Thank you for this excellent suggestion. To ensure conceptual consistency, we have standardized our terminology by shifting our preference to the term "indirect injury" throughout the entire manuscript, including the Abstract, Results, Discussion, and Figure Legends.

Comment 3: The novelty of the study could be stated more concisely, especially in relation to previous in vivo findings mentioned in the introduction.

Response: We have revised the summary of our findings at the end of the Introduction to state the novelty of this study more concisely and clearly. We emphasized that our organoid model successfully isolates direct cell-cell interactions from confounding in vivo environmental factors.

• Revised text [Lines 74-77]: "Using this new in vitro system, we successfully isolated direct podocyte-to-podocyte communication from confounding in vivo factors, such as filtration pressure and other types of cells. We demonstrate for the first time in vitro that podocyte injury propagation is an autonomous, self-sustaining process driven by direct podocyte-to-podocyte interactions."

Comment 4: The description of the organoid model and its limitations (e.g., absence of endothelial and mesangial cells) could be discussed a bit more clearly.

Response: As suggested, we have expanded the "Limitations" section in the Discussion to provide a more comprehensive and rigorous description of our organoid model. Specifically, we have explicitly stated that our organoids are derived exclusively from nephron progenitor cells (thereby lacking interstitial and ureteric bud derivatives) and have discussed how the absence of endothelial and mesangial cells represents a simplified but advantageous system that still warrants cautious interpretation regarding native diseases.

• Revised text [Lines 274-279]: "Third, our organoids are generated exclusively from nephron progenitor cells, thereby lacking components derived from interstitial progenitors and ureteric buds. Consequently, the tissue architecture of these organoids differs from that of the in vivo counterpart. While the absence of endothelial and mesangial cells allowed us to isolate and prove that they are not required for injury propagation, in native glomerular diseases, these cell types likely modulate the autonomous podocyte pathways described here."

Comment 5: Figure captions (e.g., Figs. 2 and 5) could be slightly expanded to better guide the reader in interpreting key findings such as the loss of podocin in non-targeted cells.

Response: We have expanded the legends for Figures 2 and 5 to better guide the reader in interpreting the cell-type specificity and the visual evidence of indirect injury propagation.

• Revised Fig. 2 Legend [Lines 420-421]: "...LMB2 treatment causes the loss of cluster morphology and podocyte marker expression, whereas proximal tubule structures (LTL-positive) remain unaffected, demonstrating the cell-type specificity of the immunotoxin."

• Revised Fig. 5 Legend [Lines 441-442]: "Note the complete loss of hCD25(+) podocytes (d) and the indirect reduction of podocin staining (f) in the persisting HA(+) podocytes (e), visually demonstrating the propagation of injury to non-targeted neighboring cells within the same glomerular-like structure."

Comment 6: The results section would benefit from a bit more explanation of the biological reasoning behind the observed trends.

Response: We agree with the reviewer's comment. To provide a clearer biological context for the observed gene expression trends, we have added a reference to previous in vivo findings, clarifying that the expression changes in our organoid model closely mirror those observed in chimeric mice. Additionally, we have explicitly stated the biological reasoning for targeting gap junctions in our inhibitor experiments.

The Results section has been revised as follows:

• In the Results section [Lines 202-203]: We added a clause to contextualize the gene expression changes with our previous in vivo data:

o Old: "Following LMB2 treatment, these indirectly affected podocytes displayed reduced Nphs1, Nphs2, and Wt1 expression levels and increased Gadd45b expression level."

o Revised: "Following LMB2 treatment, these indirectly affected podocytes displayed reduced Nphs1, Nphs2, and Wt1 expression levels and increased Gadd45b expression level, which is consistent with indirectly injured podocyte profiles in chimeric mice [13]."

• In the Results section [Lines 203-204]: We provided the biological rationale for examining gap junctions in the context of indirect injury propagation:

o Added text: Because it is frequently reported that gap junctions mediate bystander killing in other tissues by transferring cytotoxic molecules, we hypothesized that intercellular signaling through gap junctions might mediate the indirect injury."

Comment 7: The discussion of possible mechanisms (e.g., gap junctions, TNTs) could be presented more cautiously as potential explanations rather than definitive conclusions.

Response: We agree with the reviewer and have softened our tone in the Discussion section to present gap junctions and tunneling nanotubes (TNTs) more cautiously as potential hypotheses rather than definitive conclusions.

• Revised text for Gap Junctions [Lines 249-252]: "While these pharmacological results suggest that gap junctions may not be the principal mediators of bystander injury in this model, further genetic validation would be required to definitively exclude their contribution."

• Revised text for TNTs [Lines 263-268]: " Under such conditions, it is conceivable that injury signals may be transmitted from hCD25-positive to hCD25-negative podocytes via TNTs." …. from mosaic mice (S6 Figure). “Direct assessment of TNT involvement remains an objective for future studies.”

Comment 8: Minor typographical and formatting inconsistencies (e.g., spacing, notation, gene names) should be carefully revised.

Response: We have thoroughly reviewed the entire manuscript and corrected all typographical errors and formatting inconsistencies. Specifically, we have:

1. Corrected the gene name typo from Npsh2 to Nphs2 [Line 185].

2. Standardized the hyphenation for "2-day treatment" to match "4-day LMB2 treatment" [Line 186].

3. Unified the style for supplemental figure citations to (S2 Figure) throughout the text [Line 179].

4. Unified the style of unit (mL).

5. Corrected the spacing around the statistical symbols (e.g., p-value < 0.05) [Line 151].

6. Corrected spelling of “Schema” [Line 461].

Additional Revision by the Authors: While revising the manuscript, we noticed that the placement of the following sentences was inappropriate, given that most of the inhibitor experiments were performed using the 2-day LMB2 treatment protocol:

“Of note, suppression of Nphs1 and Nphs2 mRNAs was more pronounced after 4-day LMB2 treatment than 2-day treatment. During the 4-day treatment, the medium was changed, which washed away cell debris.”

To improve the logical flow based on our experimental timeline, we have relocated these two sentences to the second and third paragraphs of the Results section, respectively [Lines 174–175 and 185–187].

We hope that these revisions fully satisfy the requests of Reviewer #3 and that the manuscript is now acceptable for publication in PLOS ONE.

Sincerely,

Taiji Matsusaka, MD, PhD

Corresponding Author

Department of Physiology, Tokai University School of Medicine

E-mail: taijim@tokai.ac.jp

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