Urinary Biomarkers in Parkinson’s Disease: A Structured Integrative Review of Pathophysiological Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please, clarify the methods with why this is not a systematic review and a reframing as a narrative integrative review. Also, include a phrase regarding preprints being included in the manuscript. Moreover, add a short paragraph in Methods or Limitations justifying their inclusion and noting potential instability of findings.

Consider, adding a summary section about the most promising urinary biomarkers by application such as diagnosis vs progression vs stratification vs pharmacodynamics.

Check grammar, typos and abbreviation consistency. For example.: LRR2.

 

 

 

 

Author Response

1. Please clarify the methodology, explaining why this is not a systematic review study and reformulating it as a narrative integrative review. Also include a mention of the inclusion of preprints in the manuscript. Furthermore, add a brief paragraph in the Methods or Limitations section justifying the inclusion of these preprints and noting the potential instability of the results.

Reply: We have clarified the methodology as requested. The manuscript now explicitly identifies the study as a structured integrative narrative review, justifying this choice by the need to synthesize complex pathophysiological pathways rather than strictly adhering to the more restrictive parameters of a systematic review, as described in section 2.1. Study Design:

Lines 141 – 158: This study is characterized as an integrative and structured narrative review. Unlike a systematic review, which aims strictly to answer a specific clinical question through restricted parameters, this integrative approach allows the inclusion of diverse methodologies and the synthesis of complex pathophysiological pathways, providing a more holistic theoretical framework for urinary biomarkers in PD. The objective of this study is to identify and synthesize the current evidence on urinary biomarkers associated with Parkinson's disease. Given the exploratory nature of the available literature and the heterogeneity among study designs, biomarkers, and analytical significance platforms, a formal review protocol was not prospectively registered. However, the eligibility criteria, search strategy, and analytical framework were defined a priori and applied consistently throughout the review process, following selected principles from the PRISMA 2020 guidelines to increase transparency and reproducibility. Instead, the focus was on a qualitative and integrative analysis of the available evidence.

Furthermore, we have added a paragraph in the Methods section under item 2.4. Study Selection Process justifying the inclusion of preprints as a way to capture the most recent research trends, explicitly noting the limitations regarding the lack of peer review and the potential instability of their findings:

Lines 190 – 194: In order to encompass the most recent scientific advancements, pre-prints were included in this review. We acknowledge that these documents have not yet received formal peer review, which may imply a potential instability in their reported results. However, their inclusion was deemed essential to reflect the current frontier of biomarker research, and they were critically appraised alongside peer-reviewed literature.

 

2. Consider adding a summary section on the most promising urinary biomarkers by application, such as diagnosis versus progression versus stratification versus pharmacodynamics.

Reply: We thank the reviewer for this insightful suggestion, which aligns with the current regulatory and clinical frameworks for biomarker qualification. However, during our integrative analysis, we observed that the field of urinary biomarkers in PD is still in its early stages of development. Most available studies focus predominantly on diagnostic potential or pathophysiological correlation, with a significant lack of robust data regarding pharmacodynamics and stratification in urinary matrices. Due to the heterogeneity of the studies and the limited data on sensitivity and specificity across all proposed categories, we found it premature to establish a definitive classification for all these applications. However, please note that, in the Final Considerations, we discuss all possible biochemical approaches and highlight the need for integrated omics-based strategies, particularly untargeted and targeted metabolomics and peptidomics/proteomics, which emerge as promising tools for identifying combinatorial molecular signatures with improved sensitivity and specificity.

3. Check the grammar, typos, and consistency of abbreviations. For example: LRR2.

Reply: We sincerely appreciate the reviewer’s careful attention and insightful observation. We have performed a thorough linguistic and technical revision of the manuscript. The typo "LRR2" has been corrected to LRRK2 throughout the text. Regarding the abbreviation LRR, we would like to clarify that it refers to the "Leucine-Rich Repeat" domain (a specific structural motif within the protein architecture), whereas LRRK2 refers to the full enzyme (Leucine-Rich Repeat Kinase 2). Both terms are used correctly according to their respective biochemical contexts, and we have ensured that these definitions are clear in the text and figure captions to avoid any potential confusion for the reader.

 

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript entitled “Urinary Biomarkers in Parkinson’s Disease: A Structured Integrative Review Pathophysiological Pathways” by Halyne Queiroz Pantaleão Santos et al., the review about Parkinson’s disease is focused on urinary biomarkers. The text is clear, well documented, well-illustrated and highly profitable for who want to study Parkinson’s biomarker.

Major comments

• Why focus specifically on urine? The interest in urine should be presented more clearly in the abstract. The same applies to the introduction.
• L60-62: The information given regarding 12 million cases is difficult to grasp. It would be more interesting to know the incidence of the disease according to the age groups concerned, particularly the elderly.
• The method is very well described (and very interesting). However, if possible, I suggest placing all the relevant paragraphs in an appendix and keeping only one paragraph and the figure. Why? Because the subject is a review of biomarkers, not of article selection methods. If the journal does not allow appendices, it would be best to leave it as is.
• L 251: Numerical information on the gain of function would allow the reader to understand the prevalence
• L259 introduces the importance of extracellular vesicles that should be introduced earlier (see point 1)
• In Figure 3, please add arrows indicating the direction of change of the phosphorylation sites. Furthermore, the last part of the graph (the bottom part) is unclear: why are there three centrifugation steps? Why is the blood vessel shown alongside it when it is already visible in the tissues in Part 2 of the figure? I suggest modifying the graph and describing the extracellular vesicles differently.
• L302-309: Please precise the nature of the “alterations” (up or down)
• L345: Please define CNS in Abbreviations
• L349: Please define REM in Abbreviations
• Paragraph 3.1.5. If I am not mistaken, LRRK2 is associated with EV and it would make sense for it to be addressed in this paragraph, which is otherwise very short considering the importance of vesicles.
• Paragraph 3.2.2 should be associated with Figure 4 by adding the urea cycle to the figure.
• In Figure 4: The TCA cycle is drawn outside the mitochondria.
• Paragraph 34.1 should be updated with more recent article (e.g. https://doi.org/10.1002/mds.28052)
• In Figure 5, please indicate the direction of variation of the metabolite with an arrow.
• In conclusion, the essential question is which markers are relevant in urine. It can also be assumed that some are more appropriate in urine while others are present at higher concentrations in serum (and vice versa). This comparison is lacking and would be very interesting. I suggest the authors add a paragraph on this topic.
• In References, L840: unrecognized character.

Author Response

In the manuscript entitled “Urinary Biomarkers in Parkinson’s Disease: A Structured Integrative Review of Pathophysiological Pathways,” by Halyne Queiroz Pantaleão Santos et al., the review on Parkinson’s disease focuses on urinary biomarkers. The text is clear, well-documented, well-illustrated, and highly useful for anyone wishing to study biomarkers in Parkinson’s disease.

Reply: We sincerely thank the reviewer for the positive assessment of our manuscript. We believe that the subsequent suggestions provided by the reviewer significantly contributed to improving the clarity and scientific rigor of this work.

Key comments

1. Why focus specifically on urine? The interest in urine should be presented more clearly in the abstract. The same applies to the introduction.

Reply: We agree with the reviewer that the rationale for focusing on urine needed more clarity. We have updated both the Abstract and the Introduction to explicitly state the advantages of urine as a non-invasive, accessible, and biologically rich source of biomarkers that reflects systemic and central aspects of PD pathophysiology.

The following excerpt was added to the summary:

Lines 23 – 25: Unlike cerebrospinal fluid or blood, urine offers a truly non-invasive source of biomarkers, reflecting systemic metabolic changes and renal protein excretion linked to neurodegeneration.

 

The following passage was added to the introduction:

Lines 120 – 125: The focus on urinary biomarkers is justified by the clinical need for longitudinal monitoring tools that are cost-effective and patient-friendly. Furthermore, the presence of brain-derived extracellular vesicles and altered metabolites in urine provides a unique biochemical window into the pathophysiological pathways of Parkinson's Disease, bypassing the risks associated with invasive sampling.

 

2. L60-62: The information provided on 12 million cases is difficult to understand. It would be more interesting to know the incidence of the disease according to the age groups involved, particularly among the elderly.

Reply: We appreciate the reviewer's suggestion to provide more context on the incidence of the disease. We have updated the Introduction to include age-stratified prevalence data, noting that while the disease affects approximately 1% of those over 60 years of age, it reaches almost 4% in the older age groups (over 80 years), providing a clearer clinical perspective on the impact of PD on the elderly population as described in the excerpt:

Lines 62 – 68: In 2021, approximately 11.77 million people worldwide were affected by Parkinson's disease (PD). PD prevalence is highly age-dependent, affecting approximately 1% of the population over 60 years of age and rising to nearly 4% in individuals aged 80 or older. With the global aging population, these numbers are projected to double by 2040, highlighting the urgency for accessible biomarkers.

 

3. The method is very well described (and is very interesting). However, if possible, I suggest placing all relevant paragraphs in an appendix and keeping only one paragraph and the figure. Why? Because the subject is a review of biomarkers, not article selection methods. If the journal does not allow appendices, it would be better to leave it as is.

Reply: We sincerely appreciate the reviewer's suggestion and the positive feedback on the methodology. However, we have chosen to maintain the methodological description in the main text for several strategic reasons. First, as an integrative review that bridges complex pathophysiological pathways with specific urinary biomarkers, providing the full methodological context within the manuscript ensures immediate transparency and scientific reproducibility. Second, we believe that keeping this information integrated allows the reader to better understand the rigor of the study selection without the need to consult external files, which maintains the continuous flow of our scientific argument. We have, however, carefully edited the section to ensure it remains as concise and focused as possible, prioritizing the connection between the methods and the clinical findings.

 

4. L 251: Numerical information on gain of function would allow the reader to understand the prevalence.

Reply: Following the reviewer's suggestion, we have included numerical data regarding the LRRK2 G2019S mutation to provide a better understanding of its prevalence and functional impact.

Lines 278 – 282: Specifically, we added information regarding the 2-to-3-fold increase in kinase activity associated with the gain-of-function mutation and included data on the age-dependent penetrance of this variant (ranging from 28% to 74%), which clarifies the status of non-manifesting carriers mentioned in the text.

 

5. L259 introduces the importance of extracellular vesicles, which should have been introduced earlier (see point 1).

Reply: We agree with the reviewer that the importance of extracellular vesicles (EVs) should be introduced more prominently.

Lines 387 – 395 and 401 – 407 and 411 - 422: To improve the flow of the manuscript and ensure a cohesive theoretical foundation, we have restructured Section 3.1.5 Extracellular Vesicle–associated Proteins. This section now includes a more detailed introduction to the biological relevance of EVs as mediators of intercellular communication and their role in carrying pathological signatures in PD, addressing both this comment and the subsequent technical points raised regarding EVs.

 

6. In Figure 3, add arrows indicating the direction of change of phosphorylation sites. Furthermore, the last part of the graph (the bottom part) is unclear: why are there three centrifugation steps? Why is the blood vessel shown next to it, if it is already visible in the tissues in Part 2 of the figure? I suggest modifying the graph and depicting the extracellular vesicles differently.

Reply: We sincerely appreciate the reviewer's suggestion and have replaced the figure with a revised version incorporating the arrows suggested by the evaluator (please see the bottom of the figure). We also removed the blood vessels and reformulated the centrifugation steps diagram. The following sentence was added to the figure caption:

 

Lines 324  - 326: Three-step fractionation acts as a "successive filter," ensuring that what is being analyzed at the end are, in fact, the vesicles that may reflect the patient's neurodegenerative state.”

The following paragraph was also added to the text to make the importance of the three centrifugation steps in the purification process of extracellular vesicles more explicit:

Lines 388 – 395: The three-stage differential centrifugation protocol ensures rigorous isolation of extracellular vesicles (EVs) and elimination of urinary contaminants. The initial low-speed stage removes macroscopic cells and debris; the second stage eliminates protein aggregates and uromodulin polymers that can interfere with sample purity; and the final ultracentrifugation isolates the EV population of interest. This sequential approach is critical in biomarker research for Parkinson's disease, as it enables the enrichment of vesicles of neural origin while minimizing analytical interference caused by non-vesicular proteins abundant in urinary fluid.

 

7. L302-309: Please specify the nature of the "changes" (up or down)

Reply: As suggested, we have added the paragraph described below to the text, specifying the direction of the proteomic changes. We clarify that the proteins ICAM1, AHCY, and STOM showed elevated levels, while GM2A and other lysosomal enzymes showed reduced levels in GBA mutation carriers, as reported by Virreira Winter et al. (2021b).

Lines 341 – 348: In carriers of GBA mutations, significant elevations in urinary levels of proteins such as intercellular adhesion molecule 1 (ICAM1), adenosyllocysteinase (AHCY), and stomatin (STOM) were observed. In contrast, lysosomal enzymes and proteins related to glycosphingolipid metabolism exhibited divergent expression patterns compared to carriers of LRRK2 mutations. Interestingly, the reduction in levels of proteins associated with glycosphingolipid metabolism, including GM2 activator (GM2A), was more pronounced in carriers of pathogenic GBA, corroborating the role of lysosomal dysfunction in PD pathogenesis beyond classical α -synuclein aggregation [24].

 

8. L345: Please define SNC in abbreviations.

Reply: We define SNC using abbreviations as suggested.

 

9. L349: Please set REM in Abbreviations.

Reply: We define REM as an abbreviation for Rapid Eye Movement, as suggested.

 

10. Paragraph 3.1.5. If I'm not mistaken, LRRK2 is associated with EVs and it would make sense for it to be addressed in this paragraph, which, incidentally, is very short considering the importance of vesicles.

Reply: We sincerely appreciate the reviewer's suggestion and agree with relationship between LRRK2, and extracellular vesicles (EVs) is a crucial aspect. The paragraph was expanded to include the role of LRRK2 in membrane trafficking and EV secretion in the urinary context, contrasting this pathway with the metabolic and lysosomal alterations observed in DP-GBA. We added these two paragraphs to the text:

Lines 401 – 407: EVs exhibit different charges, display dynamic changes in number and content in response to physiological and environmental conditions. They can function as transporters of biomolecules, including lipids, proteins, second messengers, mRNA, miRNA, and fragments of cellular organelles, protecting them from external factors such as nucleases, proteases, and other degradative enzymes. The cargo incorporated into EVs can be transferred to target cells through ligand – receptor interaction, fusion, and/or internalization.

 

Lines 417 – 423: While DP-GBA is strongly associated with enzymatic dysfunction and substrate accumulation, DP-LRRK2 is correlated with alterations in membrane trafficking and extracellular vesicle (EV) secretion. LRRK2 is highly expressed in the renal epithelium and regulates endocytosis and autophagy processes. In carriers of pathogenic mutations, a urinary proteomic signature enriched with vesicular transport proteins and EV components has been observed, suggesting that urine may capture the impact of LRRK2 kinase hyperactivity on the endolysosomal system.

 

11. Paragraph 3.2.2 should be associated with Figure 4, adding the urea cycle to the figure.

Reply: As suggested by the reviewer, paragraph 3.2.2 was properly associated with Figure 4 (Line 468). To enrich the illustration and address the reviewer’s request, we integrated the urea cycle into the figure, representing it inside the mitochondria. We opted for an integrated schematic representation to maintain visual clarity and ensure that the complexity of the metabolic pathways presented remain understandable to the reader, avoiding excessive overlapping of graphic elements.

 

12. In Figure 4: The TCA cycle is represented outside the mitochondria.

Reply: As suggested by the reviewer, paragraph 3.2.2 was properly associated with Figure 4. To enrich the illustration and address the reviewer’s request, we integrated the TCA cycle into the figure, representing it inside the mitochondria. We opted for an integrated schematic representation to maintain visual clarity and ensure that the complexity of the metabolic pathways presented remain understandable to the reader, avoiding excessive overlapping of graphic elements.

 

13. Paragraph 34.1 should be updated with a more recent article (e.g., https://doi.org/10.1002/mds.28052 ).

Reply: The paragraph has been updated with these references to include the recent study suggested by the reviewer:

54. Liang, Y.; Zhao, Y.; Fasano, A.; Su, C.-W. Gut Permeability and Microbiota in Parkinson’s Disease: Mechanistic Insights and Experimental Therapeutic Strategies. IJMS 2025, 26, 9593, doi:10.3390/ijms26199593.
55. Kleine Bardenhorst, S.; Cereda, E.; Severgnini, M.; Barichella, M.; Pezzoli, G.; Keshavarzian, A.; Desideri, A.; Pietrucci, D.; Aho, V.T.E.; Scheperjans, F.; et al. Gut Microbiota Dysbiosis in Parkinson Disease: A Systematic Review and Pooled Analysis. Euro J of Neurology 2023, 30, 3581–3594, doi:10.1111/ene.15671.
56. Villette, R.; Ortís Sunyer, J.; Novikova, P.V.; Aho, V.T.E.; Petrov, V.A.; Hickl, O.; Busi, S.B.; De Rudder, C.; Kunath, B.J.; Heintz-Buschart, A.; et al. Integrated Multi-Omics Highlights Alterations of Gut Microbiome Functions in Prodromal and Idiopathic Parkinson’s Disease. Microbiome 2025, 13, 200, doi:10.1186/s40168-025-02227-2.
57. Chung, S.J.; Rim, J.H.; Ji, D.; Lee, S.; Yoo, H.S.; Jung, J.H.; Baik, K.; Choi, Y.; Ye, B.S.; Sohn, Y.H.; et al. Gut Microbiota-Derived Metabolite Trimethylamine N-Oxide as a Biomarker in Early Parkinson’s Disease. Nutrition 2021, 83, 111090, doi:10.1016/j.nut.2020.111090.
58. Cirstea, M.S.; Yu, A.C.; Golz, E.; Sundvick, K.; Kliger, D.; Radisavljevic, N.; Foulger, L.H.; Mackenzie, M.; Huan, T.; Finlay, B.B.; et al. Microbiota Composition and Metabolism Are Associated With Gut Function in Parkinson’s Disease. Movement Disorders 2020, 35, 1208–1217, doi:10.1002/mds.28052.

 

15. In Figure 5, indicate the direction of the metabolite's variation with an arrow.

Reply: Following the reviewer's recommendation, Figure 5 has been updated. We have added directional arrows next to each metabolite to specify the direction of the variation (increase or decrease) observed in Parkinson's Disease. This modification aims to facilitate quick reading of the figure and improve the clarity in the presentation of metabolic signatures.

 

16. In conclusion, the essential question is which markers are relevant in urine. It can also be assumed that some are more appropriate in urine, while others are present in higher concentrations in serum (and vice versa). This comparison is missing and would be very interesting. I suggest the authors add a paragraph on this topic.

Reply: We thank the reviewer for the pertinent observation. Indeed, a comparison between serum and urine biomarker profiles would enrich the discussion on the pathophysiology of the condition studied. However, the central focus of this manuscript was to conduct a specific exhaustive review of the urinary environment, aiming at non-invasive diagnostic methods; therefore, the inclusion criteria and search strategy of this study were designed exclusively to capture evidence related to urinary biomarkers. A robust comparison with serum would require a new systematic literature review, which is beyond the current scope of this research. Due to this scope limitation, we chose not to include comparative data with serum to maintain the cohesion of the work and not deviate from the main objective originally proposed.

In References, L840: unrecognized character.

Reply: We apologize for the technical error. The mentioned error appears to have been caused by a character encoding problem during PDF generation, resulting in an unreadable character on line 840. We would like to confirm that the reference in question is a classic work, essential to the theoretical foundation of this study. We have manually reviewed the citation and reference in the manuscript to ensure that all special characters have been replaced with standard formats, ensuring correct readability in this new version.