Optimization of Ibuprofen Route and Dosage to Enhance Protein-Bound Uremic Toxin Clearance During Hemodialysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Article " Optimization of Ibuprofen Route and Dosage to Enhance Protein-Bound Uremic Toxin Clearance During Hemodialysis" reflects a reality for patients undergoing dialysis. It would be interesting to relate its results to the additional treatments they receive for their systemic diseases, described in general terms.

I've outlined some aspects that I consider important for improving the article's quality. First, the abstract in the introduction should explain what ibuprofen is and what metabolic effects are attributed to it, as well as the effects of protein-bound uremic toxins. More information could be provided on the displacement of protein-bound uremic toxins from their binding sites on plasma proteins, such as the case of albumin. From a biochemical or metabolic perspective, how is the free fraction of these toxins increased to facilitate their removal during dialysis?

Regarding the results section, I find it very brief. Values for indoxyl sulfate and p-cresyl sulfate are shown, but these have not been previously linked to protein-bound uremic toxins or uremic toxins.

And why were these "dialysis parameters" and other parameters selected? What is the reason for analyzing them? Furthermore, the values presented in the tables cannot be left unaddressed.

Regarding Methodology, the methodology followed for subsequent analysis is discussed. It is assumed that the instrumentation used was previously calibrated, but nothing is said about this, nor about the standard used, nor the parameter range and/or units. If the sample was collected, it is assumed that it was placed in a tube containing a material that ensured protein stability. And when were the samples analyzed? Immediately? How many replicates were performed on the same sample? The manufacturer of the instrument, such as the LC-MS, is also mentioned. Analysis conditions are omitted, and nothing is indicated about the standard used or the chromatography phases. The exclusion time is also missing. In short, the software data and the eluent position of the analyzed proteins are lacking.

Author Response

1. Summary:

We sincerely thank the reviewers for the time devoted and for their valuable comments, which have significantly contributed to improving the quality and clarity of our manuscript. Their observations have been carefully considered and have helped strengthen both the scientific content and the presentation of the work. Below, we provide our detailed responses to each of the comments raised.

 

2. Point-by-point response to Comments and Suggestions for Authors
   1. The Article " Optimization of Ibuprofen Route and Dosage to Enhance Protein-Bound Uremic Toxin Clearance During Haemodialysis" reflects a reality for patients undergoing dialysis. It would be interesting to relate its results to the additional treatments they receive for their systemic diseases, described in general terms.

Thank you very much for your thorough review of our manuscript. With regard to the additional treatments received by patients undergoing haemodialysis, none have demonstrated, either in vitro or in vivo, a competitive or displacement effect on protein-bound uremic toxins. As the objective of this study was to evaluate the impact of different dosing regimens of a well-established displacer, namely ibuprofen, we did not include medications without a demonstrated displacement effect. According to the current literature, only five molecules have been reported to exert such a displacer effect—tryptophan, furosemide, ibuprofen, salvianolic acids, and intravenous lipid emulsions—all of which are discussed in the Discussion section (lines 175–185).

2. I've outlined some aspects that I consider important for improving the article's quality. First, the abstract in the introduction should explain what ibuprofen is and what metabolic effects are attributed to it, as well as the effects of protein-bound uremic toxins. More information could be provided on the displacement of protein-bound uremic toxins from their binding sites on plasma proteins, such as the case of albumin. From a biochemical or metabolic perspective, how is the free fraction of these toxins increased to facilitate their removal during dialysis?

First, we thank you for your comments. We have made some modifications to the abstract based on your suggestions. In order to fit the abstract into the manuscript, information about ibuprofen is found in the introduction and the discussion sections as a protein-bound uremic toxins displacer. Moreover, we have added to the manuscript at line 157, information about the displacer effect of ibuprofen which binds to the same albumin-binding site as indoxyl-sulphate and p-cresyl sulphate, becoming a competitor and increasing the serum free-fraction of indoxyl sulphate and p-cresyl sulphate. This latter effect increases the availability of these toxins, and it increases their removal during haemodialysis due to their low-molecular weight (Specifically, some studies have shown the displacer capability of ibuprofen in vitro, which also binds to Sudlow site II and therefore increases the free fraction of IS and pCS due to its higher affinity for albumin).

3. Regarding the results section, I find it very brief. Values for indoxyl sulfate and p-cresyl sulfate are shown, but these have not been previously linked to protein-bound uremic toxins or uremic toxins. And why were these "dialysis parameters" and other parameters selected? What is the reason for analyzing them? Furthermore, the values presented in the tables cannot be left unaddressed.

We thank you for your questions. As you have mentioned, we expose briefly the results of our work, highlighting the information we consider relevant for its purpose. In the introduction section, just before the results presentation, it is said that IS and pCS are two of the most studied protein-bound uremic toxins (lines 42-44). Regarding the dialysis parameters, we show the variables related to the current standard quantification of dialysis dose – total blood and KT for diffusive dose and substitution volume for convective dose – with other common variables regarding haemodialysis sessions such as initial and final weights, haematocrits, or arterial and venous pressures. The aim of this table is to demonstrate the absence of differences between haemodialysis sessions with respect to the dialysis dose, such that the differences found in the percentages of IS and pCS reduction are secondary to the different administration of ibuprofen, since this would be the only difference between the haemodialysis sessions.

4. Regarding Methodology, the methodology followed for subsequent analysis is discussed. It is assumed that the instrumentation used was previously calibrated, but nothing is said about this, nor about the standard used, nor the parameter range and/or units. If the sample was collected, it is assumed that it was placed in a tube containing a material that ensured protein stability. And when were the samples analysed? Immediately? How many replicates were performed on the same sample? The manufacturer of the instrument, such as the LC-MS, is also mentioned. Analysis conditions are omitted, and nothing is indicated about the standard used or the chromatography phases. The exclusion time is also missing. In short, the software data and the eluent position of the analysed proteins are lacking.

Thank you for your comments about the methodology of the IS and pCS measurement in the laboratory. We have added at line 258 information about the units of the IS and pCS concentrations (IS and pCS values are measured in ng/mL and reduction ratios were calculated). The technique has been developed in our institutional laboratory, and it has been validated for quantifying IS and pCS in human serum, following EMA guidelines. The method involved protein precipitation with methanol, micro-LC for chromatographic separation and detection based on accurate mass, with simultaneous high-resolution full-scan acquisition. Clinical samples from patients were obtained before and after post-dilutional hemodiafiltration session. Regarding the stability of IS and pCS in serum, the accuracy results were similar instead of the sample storage (4ºC during 24h or room temperature during 24h or after three freeze and thaw cycles. To make easy and available all the information for the lectors, we have added in the methodology the previous work in which the technique was performed and validated (More information about the biochemical methodology can be found in our previous paper (33)

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a single-center crossover study aimed at evaluating the effect of route, dose, and rate of ibuprofen administration on the dialytic removal of PBUT, specifically indoxyl sulfate (IS) and p-cresyl sulfate (pCS), during postdilution OL hemodiafiltration. The study addressed a clinically relevant and still poorly standardized issue: optimizing the use of displacers to improve PBUT clearance.

The results demonstrated that pre-filter administration of ibuprofen via the arterial line was more effective than administration via the venous line. Higher-dose arterial bolus infusion (800 mg) was associated with a higher RR of PBUT, while the lower-dose infusion of 400 mg, but at slower rate, maintained similar removal efficacy. This latter protocol may be preferred, as it may reduce systemic exposure to the drug.

The manuscript is well structured and consistent with similar findings in the literature. I suggest some considerations regarding the efficacy and safety of the proposed protocols, which should be taken into account in the discussion.

1) The authors reported an increase in RR with certain protocols compared to others, demonstrating statistical significance. However, the clinical relevance of this increase in RR was very speculative, as it remained modest in absolute terms. To give you an example, the RR of IS increased from 53.7% (baseline) to 60.8% with ibuprofen 800 mg intravenously over 2 hours. In the Discussion, to add a clinical value to their observations the Authors should correlate the observed values ​​with known toxicological thresholds.

2) The authors should specify in the Materials and Methods section whether the measurements concerned the total fraction of IS and pCS, detailing the LC/MS methodology. If this was true, as I imagine, what could be the reduction in the free fraction, i.e., the dialyzable and biologically active fraction of the PBUT?

3) The study does not provide any data on the determination of plasma ibuprofen levels (total and/or free). Therefore, the study does not provide safety data for the protocol. Although mentioned in the limitations of the study, this point needs to be adequately discussed, considering that: a) lower systemic exposure is assumed with slow infusions, and b) the dialyzability of ibuprofen depends on its protein binding and free fraction.

Author Response

We sincerely thank the reviewers for the time devoted and for their valuable comments, which have significantly contributed to improving the quality and clarity of our manuscript. Their observations have been carefully considered and have helped strengthen both the scientific content and the presentation of the work. Below, we provide our detailed responses to each of the comments raised.

 

1. Point-by-point response to Comments and Suggestions for Authors

The manuscript is well structured and consistent with similar findings in the literature. I suggest some considerations regarding the efficacy and safety of the proposed protocols, which should be taken into account in the discussion.

• The authors reported an increase in RR with certain protocols compared to others, demonstrating statistical significance. However, the clinical relevance of this increase in RR was very speculative, as it remained modest in absolute terms. To give you an example, the RR of IS increased from 53.7% (baseline) to 60.8% with ibuprofen 800 mg intravenously over 2 hours. In the Discussion, to add a clinical value to their observations the Authors should correlate the observed values ​​with known toxicological thresholds.

First of all, we thank the reviewer for his valuable commentaries about our job. Although it may seem like a modest absolute increase, we must calculate the relative difference with the baseline value. Therefore, the percentage reduction increase you exemplified would be 13.2%. Second, it is really interesting the question you bring us since it is the main question we must try to answer in the near future. It means that there are no threshold values for IS and pCS since which toxic effects appear, they might be consider as a continuous and the evidence until now suggest that the cardiovascular risk increases continuously with the increase in pCS and IS concentrations. Because of this, we have added into the Discussion the following information: “Notwithstanding, to date, no specific thresholds have been established at which toxicity related to IS and pCS becomes evident. However, the risk of cardiovascular mortality associated with these toxins correlates directly and continuously with increasing serum concentrations, both in patients with chronic kidney disease undergoing haemodialysis and in those not receiving dialysis” (line 213-217).

 

• The authors should specify in the Materials and Methods section whether the measurements concerned the total fraction of IS and pCS, detailing the LC/MS methodology. If this was true, as I imagine, what could be the reduction in the free fraction, i.e., the dialyzable and biologically active fraction of the PBUT?

Thank you for your suggestions. We have added in the methodology section that “Total IS and pCS were measured (…)” (line 273). The specific biochemical procedure is explained in our previous publication regarding this field, so in order to make it easy and available for the reader, we have added the following statement: “Total IS and pCS values were measured in ng/mL and reduction ratios were calculated. More information about the biochemical methodology can be found in our previous paper (34)”. Second, the observed reduction in the total IS and pCS in this work is mainly due to the increased availability of serum free-fraction of these toxins secondary to the displacement effect of ibuprofen. Besides, we must consider that IS and pCS albumin-bind fraction and serum free-fraction are in equilibrium, so when the free-fraction is removed by dialysis, the albumin-bind fraction is liberated in order to maintain this equilibrium, allowing its removal during the haemodialysis session.  Unfortunately, we have not analysed the differences regarding the free-fraction.

• The study does not provide any data on the determination of plasma ibuprofen levels (total and/or free). Therefore, the study does not provide safety data for the protocol. Although mentioned in the limitations of the study, this point needs to be adequately discussed, considering that: a) lower systemic exposure is assumed with slow infusions, and b) the dialyzability of ibuprofen depends on its protein binding and free fraction.

Thank you for your valuable suggestion. It is true that we assume that more prolonged and slower ibuprofen infusion rate may be accompanied with lower systemic exposure due to the combinations of its hepatic metabolism and the removal by dialysis. This hypothesis is based on the data above mentioned in the Discussion section in lines 197-199, in which a mathematical model shows a systemic exposure of 38% of the 800 mg dose administered during a 4-hour dialysis session. However, we have made some changes in the Discussion in order to make it clearer: “However, further studies specifically addressing the hypothetical differences in serum ibuprofen concentration are required as the dialyzability of ibuprofen depends on its free-fraction” (line 201-203) and “Whether a reduced likelihood of adverse events associated with ibuprofen administration in hemodialysis patients can be achieved through a more prolonged and slower infusion rate or through lower ibuprofen concentrations should be specifically evaluated in future studies” (line 211-215).

Reviewer 3 Report

Comments and Suggestions for Authors

1. Abstract: a. there is no aim/objective
2. The aim of this study 65 is to determine the best : the aim of as a study is written in past tense
3. METHOD AND MATERIAL is written after introduction and the aim of the study

Try to have subsection in method and material, such as the following:
Design, Setting, and Period of the Study

• Inclusion and Exclusion Criteria of the Sample
• Data Collection and Procedure
• Research Instrument
• Ethical Considerations
• Statistical analysis

4. Results: Provide some patients demographics, i.e age, years
5. Begin your discussion with the main results of your study!
6. provide future perspectives

• what are the bias? i.e Selection bias,  Confounding bias

7. In what way this study may help clinicians?
8. Conclusions (206-211) : must be written at the end . Before conclusion must be written Limitations of the study

 

 

Author Response

We sincerely thank the reviewers for the time devoted and for their valuable comments, which have significantly contributed to improving the quality and clarity of our manuscript. Their observations have been carefully considered and have helped strengthen both the scientific content and the presentation of the work. Below, we provide our detailed responses to each of the comments raised.

 

1. Point-by-point response to Comments and Suggestions for Authors
   1. Abstract: a. there is no aim/objective

Thank you for highlighting the absence of the aim from the study at the abstract. Following your suggestion, here is the modified version: “Background: Protein-bound uremic toxins (PBUT), particularly indoxyl sulphate (IS) and p-cresyl sulphate (pCS), are poorly removed by conventional haemodialysis because of their strong albumin binding. These toxins are associated with cardiovascular morbidity and mortality in haemodialysis patients. Displacer molecules such as ibuprofen enhance PBUT clearance by competing for albumin-binding sites, but the optimal dose and route of administration remain unclear. The aim of this study was to evaluate the effect of different ibuprofen doses, infusion durations, and routes of administration on the removal of IS and pCS during on-line hemodiafiltration (OL-HDF)”.

2. The aim of this study 65 is to determine the best: the aim of as a study is written in past tense.

Thank you for your comment. The phrase has been modified to past time: The aim of this study was to determine the best route and dosage of ibuprofen, administered during online hemodiafiltration (HDF) sessions, that maximizes PBUT clearance” (line 71-73).

3. METHOD AND MATERIAL is written after introduction and the aim of the study. Try to have subsection in method and material, such as the following:
Design, Setting, and Period of the Study

• Inclusion and Exclusion Criteria of the Sample
• Data Collection and Procedure
• Research Instrument
• Ethical Considerations
• Statistical analysis

 

Thank you very much for your suggestion in the order of the different section of the manuscript. However, we have used the template from the journal Toxins, where the Material and Method section is at the end of the template. That is the reason why this section does not appear after the Introduction section. But, following your suggestions, we have modified the “Material and Methods” section to the subsections you recommended above. Besides, we have added some information to make it clearer:

4.1 Inclusion and Exclusion criteria for the sample (line 249).

Exclusion criteria were the presence of residual kidney function, previous history of gastrointestinal bleeding, known adverse reaction to ibuprofen and treatment with other PBUT displacing molecules (loop diuretics, tryptophan, salvianolic acids, or intravenous lipid emulsions) (line 160-261).

4.2 Data Collection and Procedure (line 263)

The study followed a prospective, single-centre, crossover design. Each patient underwent nine OL-HDF sessions under different ibuprofen administration regimens (line 264-265).

4.3 Research Instrument (line 289)

Protein-bound uremic toxins (PBUT), p-cresyl sulphate (MW 108) and indoxyl sulphate (MW 213), were corrected for the degree of the haemoconcentration and the volume of distribution (approximate extracellular volume) according to Bergström and Wehle (33). Total IS and pCS were measured in serum before and after each OL-HDF session using liquid chromatography-mass spectrometry (LC-MS) (34). Total IS and pCS values were measured in ng/mL and reduction ratios were calculated. More information about the biochemical methodology can be found in our previous paper (34). Pain was assessed using the WHO visual analogue scale (VAS) before and after each session. Adverse events and treatment tolerance were recorded using structured clinical report forms and patient medical records (line 290-317).

4.4 Ethical Considerations (line 319)

All patients provided informed consent. The study was approved by the local ethic committee (HCB/2024/0173) and was conducted according to the principled of the Declaration of Helsinki (line 320-322).

4.5 Statistical analysis (line 324).

 

4. Results: Provide some patients demographics, i.e age, years

Thank you for your comment. In accordance with your suggestion, demographic data have been moved to the Results section and are now presented as baseline characteristics of the study population:

2.1 Baseline characteristics

A total of 21 patients were included with a mean age of 76.7 ± 15.8 years (66.7% women) (range 21 – 91) on a regular haemodialysis program for 52.3 ± 63.9 months (range 8 - 274). Vascular accesses were 11 native arterio-venous fistula (52.4%) and 10 tunnelled central venous catheter (47.6%). The anticoagulation used was low-molecular weight heparin in 11 patients (52.4%) and heparin sodium in 8 patients (38.1%); the remaining 2 patients had systemic anticoagulation therapy. Underlying renal diseases were nephroangioesclerosis (7 patients), diabetic nephropathy (2 patients), urologic aetiology (2 patients), interstitial nephritis (2 patients), and unknow aetiology (3 patients) (line 75-83).

 

5. Begin your discussion with the main results of your study!

Thank you for your comment. We have modified the previous paragraph for making it clearer:

This work demonstrates that administration of ibuprofen through the arterial haemodialysis line is the most effective approach to enhance IS and pCS removal during haemodialysis. In addition, prolonged and slower displacer administration achieves comparable improvements in IS and pCS clearance, which may represent an advantage by potentially reducing the risk of adverse effects related to the displacer. Additionally, our findings also indicate that lower ibuprofen doses are sufficient to significantly increase pCS removal, while higher displacer concentrations result in greater overall clearance of both toxins.

 

6. provide future perspectives

Thank you for your suggestion. We have added to the Discussion the following lines regarding the possible future perspectives:

Therefore, a more prolonged and slower administration of ibuprofen could reduce the likelihood of adverse events while simultaneously increasing the clearance of PBUT. However, further studies specifically addressing the hypothetical differences in serum ibuprofen concentration are required as the dialyzability of ibuprofen depends on its free-fraction (line 242-244).

Whether a reduced likelihood of adverse events associated with ibuprofen administration in haemodialysis patients can be achieved through a more prolonged and slower infusion rate or through lower ibuprofen concentrations should be specifically evaluated in future studies (line 253-256).

Notwithstanding, to date, no specific thresholds have been established at which toxicity related to IS and pCS becomes evident. However, the risk of cardiovascular mortality associated with these toxins correlates directly and continuously with increasing serum concentrations, both in patients with chronic kidney disease undergoing haemodialysis and in those not receiving dialysis (32). Consequently, further studies investigating molecular, endothelial, and hard outcomes concerning the impact of reducing PBUTs in haemodialysis patients are required (line 258-264).

7. what are the bias? i.e Selection bias,  Confounding bias

Thank you for your question. We have modified the paragraph regarding he limitation of the study. In fact, the main bias found in this work could be the selection bias as the sample size is relatively small and we only included patients with intradialysis pain and without kidney residual function. However, each session was developed after one week from the last session, so the possible carry-over effect was minimised. Furthermore, one of the most important limitations of our study is the absence of quantifying the ibuprofen concertation in each patient and also the necessity of adjusting for multiple comparison. So, here is the final paragraph following your comments:

This work faces certain limitations. First, the relatively small sample size restricts, and the single-centre design may limit its generalizability. In addition, the necessity of adjusting for multiples comparison through stringent statistical thresholds might have underpowered the detection of smaller, albeit clinically relevant, differences between infusion rates. Second, we only assessed IS and pCS, limiting the extrapolation of these results to other not measured PBUT. Third, serum ibuprofen concentrations were not measured, preventing experimental confirmation of the predictions derived from the above-mentioned mathematical model. However, this is the first work that compares different displacer administration sources to evaluate their efficacy in increasing PBUT removal in patients on regular haemodialysis treatment.

 

8. In what way this study may help clinicians?

Thank you for your interesting question. This question is the main we must try to answer. The first thing to do is finding the best biomarker of endothelial function or the cardiovascular system in order to assess its changes through PBUT kinetics. However, at the present moment, there is no such biomarker available. Nonetheless, there is enough evidence until our days that demonstrates the link between indoxyl-sulphate and p-cresyl sulphate with cardiovascular event in patients with chronic kidney disease in haemodialysis and with no-dialysis; so, every clinically available strategy that diminish PBUT concentration may be useful for clinicians.   

9. Conclusions (206-211) : must be written at the end . Before conclusion must be written Limitations of the study

Thank you for your comment. We have made some changes of the work, so the conclusions are presented at the end of the Discussion section. Additionally, a paragraph addressing the study's limitations has been included immediately preceding the conclusion.

Reviewer 4 Report

Comments and Suggestions for Authors

Protein-bound uremic toxins (PBUT), indoxyl sulphate (IS) and p-cresyl sulphate (pCS) are generated through an initial  metabolism by the gut microbiota, followed by hepatic metabolism. pCS is considered the most toxic one with the highest toxicity score with current experimental and clinical data, affecting up to seven different organs. This work shows that the arterial haemodialysis line is the most effective site for ibuprofen infusion to improve IS and pCS removal during haemodialysis. Prolonged and slower displacer administration is equally effective in enhancing IS and pCS clearance,  which could be advantageous in reducing the clinical adverse effects associated with the  displacer. This is a prospective study done in a single centre with 21 patients.

The Introduction is well written providing information about the need of the study.

The Materials and methods are disclosed completely and the flow of the article is also very good.

The results are well explained and the relevant tables and figures portray the findings of the study.

However, I have a few minor comments:

1. The sample size is too small. I suggest that the authors should mention the limitations of the study.
2. The age of the patients were around 76 +_ 15 years. Can there be any other co0morbidity associated at this age that can increase the level of these PBUT? In other words, the generation and clearance of PBUT from blood is same or different for the different patient group. Please explain.

Minor comments

The English language needs minor editing. I found a few mistakes, many more can be present. Kindly revisit the entire manuscript for English language corrections.

 

Line # 75: "Pain after dialysis was significantly lower independently the posology". The sentence is meaningless. Please rectify.

Line #79: "There were no differences  between dialysis sessions.". Do the authors mean, no significant difference?

 

I recommend a major revision.

Author Response

We sincerely thank the reviewers for the time devoted and for their valuable comments, which have significantly contributed to improving the quality and clarity of our manuscript. Their observations have been carefully considered and have helped strengthen both the scientific content and the presentation of the work. Below, we provide our detailed responses to each of the comments raised.

1. Point-by-point response to Comments and Suggestions for Authors

The Introduction is well written providing information about the need of the study. The Materials and methods are disclosed completely, and the flow of the article is also very good. The results are well explained, and the relevant tables and figures portray the findings of the study.

However, I have a few minor comments:

1. 1. The sample size is too small. I suggest that the authors should mention the limitations of the study.

Thank you very much for your comments. We have modified the previous paragraph regarding the limitation of the study. In the new one, we hope that they were presented much clearer:

This work faces certain limitations. First, the relatively small sample size restricts, and the single-centre design may limit its generalizability. In addition, the necessity of adjusting for multiples comparison through stringent statistical thresholds might have underpowered the detection of smaller, albeit clinically relevant, differences between infusion rates. Second, we only assessed IS and pCS, limiting the extrapolation of these results to other not measured PBUT. Third, serum ibuprofen concentrations were not measured, preventing experimental confirmation of the predictions derived from the above-mentioned mathematical model. However, this is the first work that compares different displacer administration sources to evaluate their efficacy in increasing PBUT removal in patients on regular haemodialysis treatment.

 

2. The age of the patients were around 76 +_ 15 years. Can there be any other co0morbidity associated at this age that can increase the level of these PBUT? In other words, the generation and clearance of PBUT from blood is same or different for the different patient group. Please explain. 

Thank you for your insightful question. We are mindful of the advanced age of our cohort and the likely higher prevalence of comorbidities associated with both aging and chronic kidney disease (CKD). However, within our dialysis unit, we have not observed significant differences in the baseline concentrations of IS and pCS, nor in their reduction ratios, based on age or comorbidities. Nevertheless, in a previously published study, we did find differences in the reduction ratios of these toxins when analysed by sex (Escudero-Saiz, V. J., Factors influencing the removal of protein-bound uremic toxins in hemodiafiltration. Nefrologia, 45(10), 501391. https://doi.org/10.1016/j.nefroe.2025.501391). It is well-established that CKD is associated with alterations in the gut microbiota, promoting an overgrowth of proteolytic bacteria over saccharolytic species, as well as a decrease in butyrate-producing species—findings similar to those observed in inflammatory bowel disease—which promote protein-bound uremic toxins generation. However, none of the patients included in our study presented relevant gastrointestinal diseases. Another point that may be relevant is the kind of diet followed by the patients, as the Medika study demonstrated that a mediterranean diet or a very-low protein diet reduced the concentration of different PBUT compared to a western diet; the authors discussed these results mainly for two reasons: first, the reduction in the protein intake may reduce the availability of the PBUT precursors to the microbiota, and, second, these diet modification changed the composition of the gut microbiota, increasing those saccharolytic species mentioned above. However, all our patient follow wester or mediterranean diet but there were no records of this information.

 

Minor comments

The English language needs minor editing. I found a few mistakes, many more can be present. Kindly revisit the entire manuscript for English language corrections.

Line # 75: "Pain after dialysis was significantly lower independently the posology". The sentence is meaningless. Please rectify.

Thank you for your comment. We change the phrase into the next one: A significant reduction in pain was observed post-dialysis, irrespective of the ibuprofen regimen administered (line 91-92).

Line #79: "There were no differences  between dialysis sessions.". Do the authors mean, no significant difference?

Thank you for your suggestion. In order to make it clearer, we have changed the phrase into this one: Dialysis parameters remained consistent across all study arms, with no statistically significant variations detected (line 99-101).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Having reviewed the new version, I believe the authors have improved the presentation of their results regarding ibuprofen administration in patients undergoing hemodialysis. The article can be published.

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further comments

Reviewer 3 Report

Comments and Suggestions for Authors

NO MORE

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed all the queries raised by me. The article can be accepted for publication.