Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer comments

Summary
This is a well-structured and timely review that connects embryologic programs with molecular pathways and clinical genetics in CAKUT. The manuscript effectively integrates developmental signaling (GDNF–RET, WNT/β-catenin, BMP/FGF), ciliogenesis/PCP, and ECM biology with monogenic, CNV, and multigenic etiologies, and highlights emerging tools (organoids, gene editing, single-cell/spatial omics) with clear clinical relevance.

Minor Revision:
When discussing genomic sequencing technologies, including whole-exome sequencing (WES), whole-genome sequencing (WGS), and copy-number variant (CNV) analysis, the authors should clarify the distinctions among these methods. Specifically, CNVs can be detected using both WES and WGS platforms. The authors are encouraged to cite relevant literature, such as “Progress and clinical prospect of genomic structural variants investigation” (PMID: 38310047), to illustrate how third-generation sequencing technologies may further advance the detection of structural variants and their clinical significance for CAKUT in the future.

Author Response

Comment: When discussing genomic sequencing technologies, including whole-exome sequencing (WES), whole-genome sequencing (WGS), and copy-number variant (CNV) analysis, the authors should clarify the distinctions among these methods. Specifically, CNVs can be detected using both WES and WGS platforms. The authors are encouraged to cite relevant literature, such as “Progress and clinical prospect of genomic structural variants investigation” (PMID: 38310047), to illustrate how third-generation sequencing technologies may further advance the detection of structural variants.

Response: We thank the reviewer for this helpful comment. We have revised the manuscript to clarify the distinctions among WES, WGS, and CNV analysis, explicitly noting that CNVs can be detected using both WES- and WGS-based approaches, with differences in genomic coverage and sensitivity. In addition, we expanded the discussion to include the potential role of third-generation long-read sequencing technologies in improving the detection and characterization of structural variants relevant to CAKUT. These changes have been incorporated in the revised manuscript (Introduction section, page 2, lines 44–52), and the suggested reference has been added to the bibliography (Ref. No. 7).

Reviewer 2 Report

Comments and Suggestions for Authors

In their review titled "From genes to malformations: molecular mechanisms driving the pathogenesis of congenital anomalies of the kidney and urinary tract" Fourikou and Dotis have described the molecular foundations of kidney development, pathways and mechanisms underlying CAKUT, the genetic etiology of CAKUT, epigenetic and environmental influences, and integrative approaches for clinical care and research of CAKUT. Their work presents a valuable contribution to the field and I only have a few very minor comments and suggestions about improving the manuscript.

1)  Lines 65-70: I suggest adding a statement about the usefulness of human embryonic kidney tissue samples which, although rare, can be further evidence that the identified CAKUT candidate genes and their respective proteins are indeed expressed during normal human nephrogenesis. An examples would be the work by Westland et al. (doi: 10.1038/ki.2015.239).

2) Lines 264-269: These statement are already mentioned in prior sections of the manuscript and can be removed as no new information is given.

3) Line 313: The names of genes should be written in italics. Please make appropriate changes throughout the manuscript when referring to genes.

 

Author Response

Comment 1: Lines 65–70: I suggest adding a statement about the usefulness of human embryonic kidney tissue samples which, although rare, can be further evidence that the identified CAKUT candidate genes and their respective proteins are indeed expressed during normal human nephrogenesis.

Response: We thank the reviewer for this insightful suggestion. We have added a statement emphasizing the value of human embryonic kidney tissue studies as direct evidence that several CAKUT candidate genes and their corresponding proteins are expressed during normal human nephrogenesis, thereby strengthening the biological relevance of findings derived from experimental and in vitro models. This addition has been incorporated into the revised manuscript (page 2, lines 78–82), and the corresponding reference has been included [Ref. 22].

Comment 2: Lines 264–269: These statements are already mentioned in prior sections of the manuscript and can be removed as no new information is given.

Response: We agree with the reviewer that the statements in lines 264–269 were repetitive and did not provide additional information beyond what was already discussed in earlier sections of the manuscript. To improve clarity and avoid redundancy, we have removed this text from the revised manuscript. This change has been implemented in the revised version (page 7).

Comment 3: Line 313: The names of genes should be written in italics. Please make appropriate changes throughout the manuscript when referring to genes.

Response: We thank the reviewer for pointing out this formatting issue. Gene symbols have now been consistently formatted in italics throughout the entire manuscript, in accordance with standard genetic nomenclature conventions. This includes all occurrences in the main text, tables, and figure legends, while protein names and pathway components remain in upright font. The necessary corrections have been implemented in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations to the authors for their work. This is a comprehensive up to date review of  the molecular and develpmental mechanism underlying CAKUT. The manuscript is well structured and scientifically sound.

Some things to consider -  some concepts such as UB–MM interactions, GDNF–RET signaling, and the multifactorial nature of CAKU are repeated across multiple sections.

Perhaps highlighting unresolved controversies, gap in the current knowledge or limitations of existing studies would enhance the article.

Overall, this is a strong review that will be of interest to both basic scientists and clinicians working in pediatric nephrology, genetics, and developmental biology. With minor revisions focused on synthesis and conciseness , the manuscript would be well suited for publication.

 

Author Response

Comment: Some concepts such as UB–MM interactions, GDNF–RET signaling, and the multifactorial nature of CAKUT are repeated across multiple sections. Perhaps highlighting unresolved controversies, gaps in current knowledge or limitations of existing studies would enhance the article.

Response: We appreciate this insightful comment. In response, we have revised the manuscript to reduce repetition of core concepts, including UB–MM interactions, GDNF–RET signaling and the multifactorial nature of CAKUT, by removing redundant statements across sections. Specifically, repetitive text has been deleted to improve narrative flow and conciseness (old version page 7, lines 264–269 deleted). In addition, we have strengthened the integrative and forward-looking aspects of the review by explicitly highlighting current gaps in knowledge and limitations of existing studies. A dedicated paragraph addressing unresolved issues, including incomplete genetic diagnostic yield, challenges in interpreting noncoding and structural variants, variability in genotype–phenotype correlations, and limitations of experimental and in vitro models in fully recapitulating human nephrogenesis, has been added to Section 8.4 (page 12, lines 487-496). Finally, the Conclusions have been revised to integrate these limitations and emphasize future research directions that address current knowledge gaps through combined genomic, experimental and clinical approaches (page 12, lines 516-521).

Reviewer 4 Report

Comments and Suggestions for Authors

This paper presents a thorough and organized review of the molecular, genetic, epigenetic, and environmental mechanisms implicated in CAKUT. With the combination of embryological insights with contemporary advances in genomics, multi-omics technology, and experimental modelling techniques, the review is both timely and relevant. The paper boasts a reference list that is admirably wide-ranging and up-to-date, with numerous references from 2024-2025.

In general, this article can be considered useful to basic scientists and clinicians interested in pediatric nephrology, developmental biology and medical genetics. With some minor editing, it can be considered for publication, with clarification, balance, and a focus on certain sections of the article.

1. Keep a balance between depth and redundancy, e.g. GDNF-RET signalling, WNT/β-catenin, and ciliary dysfunction, for example, are some pathophysiological mechanisms mentioned often.
2. Where appropriate, sections should be consolidated or cross-references should be made for the reduction of duplication.
3. Figure 1 does not mean anything clear from the text. A more wide-ranging figure legend or brief description might be warranted.
4. It would be helpful to have a schematic including embryologic stages, disrupted pathways, and CAKUT phenotypes in one image.
5. Though informative, Section 5 is shorter and less mechanistic than the sections on genetics and genomics.
6. More information about the interaction of certain exposures (e.g. retinoic acid-WNT, vitamin D-BMP) and known signalling pathways would improve this section.
7. The application of precision treatments remains largely theoretical. Some balance could have been achieved by a brief mention of the barriers, ethics, and feasibility of targeting developmental pathways in utero or soon after birth.
8. The Conclusions section also contains a numbering error; it labels the section with "5. Conclusions.
9. Errors in the original document, such as repeated text (e.g. lines 263 to 268), need correction.
10. A non-specialist reader often struggles to follow the list of abbreviations and the density of abbreviations. This is especially true when they are the same.
11. Tables 1-4 are informative. Short captions would help them. These captions should have one or two sentences. They should discuss the data's clinical or biological importance.                                                                                                                                                          To sum up, the manuscript is scientifically solid, well-written, and highly informative. Addressing the points above—particularly minor redundancies, editorial adjustments, and slight expansion of epigenetic and translational discussions—will further improve clarity and impact. I recommend acceptance following minor revisions.

Author Response

Comment 1: Keep a balance between depth and redundancy, e.g. GDNF–RET signalling, WNT/β-catenin, and ciliary dysfunction are some pathophysiological mechanisms mentioned often.

 

Response: We thank the reviewer for this important observation. In response, we have carefully revised the manuscript to reduce redundancy while preserving the necessary depth for key developmental mechanisms, including GDNF–RET signaling, WNT/β-catenin pathways and ciliary dysfunction. Repetitive descriptions of these core concepts across sections have been removed or streamlined, particularly where similar statements were reiterated without adding new mechanistic or clinical insight. These revisions improve conciseness and narrative flow while maintaining the conceptual framework required for a comprehensive understanding of CAKUT pathogenesis. Specifically, redundant text has been deleted from the revised manuscript (old version page 7, lines 264–269).

Comment 2: Where appropriate, sections should be consolidated or cross-references should be made for the reduction of duplication.

Response: We thank the reviewer for this suggestion. In response, we have consolidated overlapping content across sections by removing duplicated text and streamlining related paragraphs, rather than introducing explicit cross-references that could interrupt narrative flow. This approach allowed us to reduce redundancy while preserving the logical progression of concepts and maintaining readability. These changes have been implemented throughout the revised manuscript, contributing to improved coherence and conciseness.

Comment 3: Figure 1 does not mean anything clear from the text. A more wide-ranging figure legend or brief description might be warranted.

Response: We thank the reviewer for this helpful comment. To improve clarity and ensure that Figure 1 is more readily interpretable in the context of the manuscript, we have revised and expanded the figure legend to provide a clearer description of the genetic mechanisms depicted and their relevance to CAKUT pathogenesis. The updated legend now explicitly explains the relative contributions of monogenic, copy-number and multigenic architectures and their convergence on key developmental pathways. These changes have been implemented in the revised manuscript (Figure 1 legend)

Comment 4: It would be helpful to have a schematic including embryologic stages, disrupted pathways, and CAKUT phenotypes in one image.

Response: We agree that an integrated schematic linking embryologic stages, disrupted signaling pathways and CAKUT phenotypes would be valuable. However, given the scope of this review and the breadth of developmental processes covered, we elected to retain the existing figures and tables rather than introduce an additional schematic figure. Instead, we strengthened conceptual integration across the manuscript by expanding the legend of Figure 1 and by enhancing the captions of Tables 1–4, which collectively summarize developmental stages, molecular mechanisms, genetic architectures, prenatal imaging findings and representative CAKUT phenotypes. We believe this approach provides an integrated conceptual framework while preserving clarity and avoiding redundancy and excessive figure complexity.

Comment 5: Though informative, Section 5 is shorter and less mechanistic than the sections on genetics and genomics.

Response: In response to the reviewer’s comment, we have strengthened the mechanistic content of this section by explicitly describing how maternal and environmental exposures interact with core developmental signaling pathways. Specifically, we added a mechanistic link between retinoic acid and vitamin D signaling and key pathways such as WNT/β-catenin and BMP, highlighting their influence on ureteric bud patterning, nephron progenitor maintenance and susceptibility to CAKUT. These revisions improve balance across sections while remaining consistent with the available level of mechanistic evidence (Section 5.3, page 8, lines 324–329).

Comment 6: More information about the interaction of certain exposures (e.g. retinoic acid–WNT, vitamin D–BMP) and known signalling pathways would improve this section.

Response: In response, we expanded Section 5.3 to describe how environmental exposures interact with core developmental signaling pathways. Specifically, we added mechanistic examples showing how retinoic acid and vitamin D signaling modulate WNT/β-catenin and BMP pathways, influencing ureteric bud patterning and nephron progenitor maintenance (Section 5.3, page 8, lines 324–329).

Comment 7: The application of precision treatments remains largely theoretical. Some balance could have been achieved by a brief mention of the barriers, ethics, and feasibility of targeting developmental pathways in utero or soon after birth.

Response: We thank the reviewer for this valuable point. In response, we have added a brief discussion in Section 8.4 addressing the limitations, feasibility challenges, and ethical considerations of targeting developmental signaling pathways, particularly in prenatal or early postnatal settings. We emphasize that most precision treatments for CAKUT remain theoretical and face significant biological, technical, and ethical barriers. These additions offer a more realistic view of the translational potential (Section 8.4, page 12, lines 487–496).

 

Comment 8: The Conclusions section also contains a numbering error; it labels the section with “5. Conclusions”.

Response: We thank the reviewer for pointing out this error. The section numbering has been corrected in the revised manuscript.

Comment 9: Errors in the original document, such as repeated text (e.g. lines 263–268), need correction.

Response: We thank the reviewer for identifying this issue. The repeated text has been removed from the revised manuscript to improve clarity and avoid redundancy (old version page 7, lines 264–269).

Comment 10: A non-specialist reader often struggles to follow the list of abbreviations and the density of abbreviations.

Response: We thank the reviewer for this helpful comment. In response, we have made targeted revisions to improve readability for non-specialist readers by reducing abbreviation density in selected sentences. Specifically, we revised gene-dense passages to improve clarity and spelled out clinical terms at first mention (e.g., vesicoureteral reflux), while preserving the use of standard gene symbols and scientific precision. These changes enhance accessibility without altering the technical content of the review (page 7, lines 245-249).

Comment 11: Tables 1–4 are informative. Short captions would help them. These captions should have one or two sentences and discuss the data’s clinical or biological importance.

Response: We thank the reviewer for this helpful suggestion. In response, we have revised the captions of Tables 1–4 to include concise one- to two-sentence descriptions highlighting their biological and clinical relevance, thereby ensuring that each table is self-contained and easily interpretable.