Nitric Oxide Synthesis Metabolites—As Potential Markers in Chronic Kidney Disease in Children

Round 1

Reviewer 1 Report

The authors analyzed the NO synthesis metabolites ADMA, SDMA, DMA and citrulline in the plasma of children with CKD using plasma liquid chromatography-mass spectrometry. A significant improvement especially regarding the presentation of the data and therefore their interpretation is needed to make this manuscript acceptable for publication.

Abstract

Lines 20-22: It is stated that “There were significant differences observed in levels of ADMA, SDMA, DMA and citruline between groups within different stages of CKD (p ˂0.001).” However, Table 3 shows a p-value of 0.001 only for ADMA.

Line 21: citrulline instead of citruline

There is no conclusion at the end of the abstract!

Introduction

Lines 45 ff.: It would be useful if the reactions described in this paragraph were also represented in a figure.

Materials and Methods

Table 1:

“…and control group” should be added to the title “The biochemical characteristics of the blood of studied patients population.

It should be explained/commented why N is smaller than 48 or 33 for some parameters, and especially why N is 34 for Hb, Ht and RBC in the control group.

Figure 2:

The data presented in this figure is incomprehensible considering that the authors used the same methodology as in reference [23] where completely different results (retention times) are shown (Figure 1 in [23]).

The retention times of the main peaks of citrulline and arginine are the same!

In the chromatogram of the patient sample there are no peaks corresponding to the metabolites investigated.

Results

Table 2: N should be shown separately for the study and control group.

Table 3: The presentation of this table should be made clearer.

Figure 3: Considering that the data shown in this figure is the same as in table 3, the same order of metabolites would be useful.

Table 4: The data shown here are not described in the text.

Figure 4: This figure is not mentioned in the text. There is no appropriate explanation in the text or in the legend.

Discussion

It would be helpful if reference was made to the respective table or figure when discussing specific data.

Some references are not in the correct order (25, 38 and 39) and reference 26 does not appear in the text.

Lines 247-250: This sentence should be rephrased for clarity.

Line 273: Snauwaert et al. is a Belgian and not a German research team.

Lines 277-278: It is stated that “Plasma ADMA levels do not correlate with blood creatinine or eGFR levels…” However, a significant correlation is shown in table 4. This should be clarified.

Lines 343-344: It is stated that “In our study, citrulline correlated statistically significantly positively only with urea.” However, this contradicts data shown in table 4.

The authors analyzed the NO synthesis metabolites ADMA, SDMA, DMA and citrulline in the plasma of children with CKD using plasma liquid chromatography-mass spectrometry. A significant improvement especially regarding the presentation of the data and therefore their interpretation is needed to make this manuscript acceptable for publication.

Abstract

Lines 20-22: It is stated that “There were significant differences observed in levels of ADMA, SDMA, DMA and citruline between groups within different stages of CKD (p ˂0.001).” However, Table 3 shows a p-value of 0.001 only for ADMA.

Line 21: citrulline instead of citruline

There is no conclusion at the end of the abstract!

Introduction

Lines 45 ff.: It would be useful if the reactions described in this paragraph were also represented in a figure.

Materials and Methods

Table 1:

“…and control group” should be added to the title “The biochemical characteristics of the blood of studied patients population.

It should be explained/commented why N is smaller than 48 or 33 for some parameters, and especially why N is 34 for Hb, Ht and RBC in the control group.

Figure 2:

The data presented in this figure is incomprehensible considering that the authors used the same methodology as in reference [23] where completely different results (retention times) are shown (Figure 1 in [23]).

The retention times of the main peaks of citrulline and arginine are the same!

In the chromatogram of the patient sample there are no peaks corresponding to the metabolites investigated.

Results

Table 2: N should be shown separately for the study and control group.

Table 3: The presentation of this table should be made clearer.

Figure 3: Considering that the data shown in this figure is the same as in table 3, the same order of metabolites would be useful.

Table 4: The data shown here are not described in the text.

Figure 4: This figure is not mentioned in the text. There is no appropriate explanation in the text or in the legend.

Discussion

It would be helpful if reference was made to the respective table or figure when discussing specific data.

Some references are not in the correct order (25, 38 and 39) and reference 26 does not appear in the text.

Lines 247-250: This sentence should be rephrased for clarity.

Line 273: Snauwaert et al. is a Belgian and not a German research team.

Lines 277-278: It is stated that “Plasma ADMA levels do not correlate with blood creatinine or eGFR levels…” However, a significant correlation is shown in table 4. This should be clarified.

Lines 343-344: It is stated that “In our study, citrulline correlated statistically significantly positively only with urea.” However, this contradicts data shown in table 4.

Author Response

Dear Reviewer,

Thank you for your apt remarks giving us the opportunity to improve our manuscript.

Abstract: Lines 20-22: It is stated that “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between groups within different stages of CKD (p ˂0.001).” However, Table 3 shows a p-value of 0.001 only for ADMA.

Answer: Sentence was corrected to “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between  control  vis CKD groups ( p<0.001-p<0.029).  “

Line 21: citrulline instead of citruline

Answer: This is corrected.

There is no conclusion at the end of the abstract!

Answer: Following passage was added: “Children with CKD develop disturbances in most metabolites of NO synthesis. Dialysis  children treated  show the greatest disturbances  of plasma ADMA and citrulline levels. ADMA seems to be a good indicator of the gradual progression of the CKD, which is proved by the negative correlation with eGFR”

Introduction

Lines 45 ff.: It would be useful if the reactions described in this paragraph were also represented in a figure.

Answer: New diagram showing reactions was made.

Materials and Methods

Table 1: “…and control group” should be added to the title “The biochemical characteristics of the blood of studied patients population.

Answer: This is completed as suggested

It should be explained/commented why N is smaller than 48 or 33 for some parameters, and especially why N is 34 for Hb, Ht and RBC in the control group.

Answer:  Data used in study is based on patients medical documentation. Sadly it was not always complete. Lack of data in some parameters is a result of incomplete patient documentation.

We added adequate information in the materials and methods section.

Figure 2: The data presented in this figure is incomprehensible considering that the authors used the same methodology as in reference [23] where completely different results (retention times) are shown (Figure 1 in [23]).

Answer: due to the presentation of method in [23] and with DMA in [24] we have shown here the chromatograms for representative patient sample, namely, the extracted current chromatograms for each individual compound benzamide derivative and at the bottom the total ion current chromatogram for the sample. The amount of each compound in sample was calculated from calibration curves prepared for each compound. The retention times are different, of ADMA is 215 and SDMA is 235 sec, so they are clearly separated. Therefore it was avoid showing repeated picture from the methodological papers [23, 24], instead the presented are data from individual patient to present the way how compounds were separated and quantitated. So, all chromatograms are from the patient sample, but individual compounds are shown as selected ion chromatograms and the bottom chromatogram is total ion current chromatogram. Selected ion chromatograms enable to quantitate compounds present in noise of total ion chromatogram. The text in legend is corrected and more clear in revised version of manuscript.   

Results

Table 2: N should be shown separately for the study and control group.

Answer: N was added

Table 3: The presentation of this table should be made clearer.

Table construction was based on CIMB template. Changes were made so the reading of table is easier.

Figure 3: Considering that the data shown in this figure is the same as in table 3, the same order of metabolites would be useful.

Answer: This is corrected.

Table 4: The data shown here are not described in the text. Figure 4: This figure is not mentioned in the text. There is no appropriate explanation in the text or in the legend.

We merged figure 4 and table 4. The figure legend was extended and adequate references were added in the text.

Discussion

It would be helpful if reference was made to the respective table or figure when discussing specific data.

Adequate references were added.

Some references are not in the correct order (25, 38 and 39) and reference 26 does not appear in the text.

Order of references was corrected

Lines 247-250: This sentence should be rephrased for clarity.

Answer: The sentence is improved: Similar results were found for the arginine value; our CKD children had the lower levels (without statistical significance compared to the control) of arginine values in the CKD children, like were (statistically significantly lower) in El-Sadek AE et al. [11].

Line 273: Snauwaert et al. is a Belgian and not a German research team.

Answer: the sentence is corrected.

Lines 277-278: It is stated that “Plasma ADMA levels do not correlate with blood creatinine or eGFR levels…” However, a significant correlation is shown in table 4. This should be clarified.

Answer:  This original sentence is :

“Plasma ADMA levels do not correlate with blood creatinine or eGFR levels in children and adolescents with CKD, most likely because most ADMA is enzymatically degraded in the opinion of Hsu Chien-Ning et al. [34]”

In our observations shown that plasma  ADMA correlated positively with creatinie , and  negatively with  eGFR ( Table 4 ), which we mention in the following lines in manuscript.

Lines 343-344: It is stated that “In our study, citrulline correlated statistically significantly positively only with urea.” However, this contradicts data shown in table 4.

Answer: the sentence is corrected: “In our study, citrulline correlated statistically significantly positively with urea, ADMA, SDMA and DMA.”

Reviewer 2 Report

The authors reported the positive correlations of statistical significance were found between: ADMA and creatinine, SDMA, DMA and citrulline. Along with the progression of CKD, there was to increase plasma concentrations of ADMA, SDMA, DMA, citrulline and arginine, although not always of statistical significance. The highest concentration values were demonstrated in the group of RRT except for such metabolites as SDMA and arginine. At the pre-dialysis stage of CKD, a sharp decrease in analyte concentration (arginine, DMA, citrulline) was observed.

However, some concerns have been raised.

1.     The aim of Current Issues in Molecular Biology is a peer-reviewed journal publishing original articles and reviews in all areas of molecular biology. However, this paper did not find any molecular biology in it and suggest to submit to clinical medicine in pediatric area.

2.     Lack of detail of basal and biochemical characteristics data in Table between different groups.

3.     Many factors may affect NO synthesis, such as drugs (proton pump inhibitors, rosiglitazone, pioglitazone, amlodipine, valsartan, perindopril, estrogen, statins…), albuminuria or proteinuria, urine output, comorbidity and did not mention in this study.

4.     ADMA is excreted mainly by metabolism by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2, and to a lesser degree by alanine glyoxylate aminotransferase 2 (AGTX2), butylation, and methylation, while renal excretion represents a secondary and less important route of excretion (< 20%). SDMA’s major route of elimination is renal excretion (>90%), with metabolism being a minor route of elimination. How to explain this finding of this study that ADMA is negatively associated with GFR and SDMA is not associated with GFR?

5.     Did not mention about limitation of this study.

Author Response

Dear Reviewer,

Thank you for your apt remarks giving us the opportunity to improve our manuscript.

1. The aim of Current Issues in Molecular Biology is a peer-reviewed journal publishing original articles and reviews in all areas of molecular biology. However, this paper did not find any molecular biology in it and suggest to submit to clinical medicine in pediatric area.

Answer : This journal was recommended to us by assistant of MDPI as suitable for our work. Even though we didn’t use classical methods of molecular biology such as PCR or gene sequencing, we see our work relevant to scope of the journal. There is no clear demarcation line between molecular biology and biochemistry, our study involves topic such as metabolomics and use of spectroscopy which are connected to molecular biology. We don’t see clinical importance as a disadvantage.

Several articles regarding oxidative stress were published in CIMB i.e. https://doi.org/10.3390/cimb44050124 , https://doi.org/10.3390/cimb44030091, as well as several articles form clinical areas such as dentistry medicine https://doi.org/10.3390/cimb44030085 or cardiology https://doi.org/10.3390/cimb44030092.

Furthermore this manuscript was assigned to Section “Bioorganic Chemistry and Medicinal Chemistry” in Special Issue “Bioactive Compounds of Natural Products on Metabolic Disorders and Complications” which are domains appropriate for our work.

2. Lack of detail of basal and biochemical characteristics data in Table between different groups.

Answer : We added table with clinical and biochemical characteristics of patients in different stages of CKD in the supplementary materials.

3. Many factors may affect NO synthesis, such as drugs (proton pump inhibitors, rosiglitazone, pioglitazone, amlodipine, valsartan, perindopril, estrogen, statins…), albuminuria or proteinuria, urine output, comorbidity and did not mention in this study.

Answer : Standard therapeutic procedures were used as described in the materials and methods. We are aware that drugs affect the level of ADMA and SDMA.

Further studies on more selective groups would allow for a better assessment of the effect of specific drugs and clinical situations. In-depth multivariate analysis would be impossible with the number of patients we have. Adequate information was added to limitations of the study.

4. ADMA is excreted mainly by metabolism by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2, and to a lesser degree by alanine glyoxylate aminotransferase 2 (AGTX2), butylation, and methylation, while renal excretion represents a secondary and less important route of excretion (< 20%). SDMA’s major route of elimination is renal excretion (>90%), with metabolism being a minor route of elimination. How to explain this finding of this study that ADMA is negatively associated with GFR and SDMA is not associated with GFR?

Answer : There are several paths that can lead to increased levels of ADMA and SDMA:

1) increased oxidative stress;

2) disruption of renal excretion of metabolite;

3) disruption of function of the endothelium.

In CKD we observe presence of multiple factors influencing both synthesis and excretion of SDMA and ADMA. Thus there are several explanations why  “ADMA is negatively associated with GFR and SDMA is not associated with GFR” it is plausible that levels ADMA is more sensitive to oxidative stress and endothelial dysfunction caused by impaired renal function which can be measured by GFR.

In our research results: both ADMA and SDMA show a negative correlation with eGFR, but in the case of ADMA it is statistically significant, while for SDMA it is not so significant. There is also plausibility of type II error, after taking into account relatively small sample group.

ADMA metabolism by DDAH 1 and 2 isoforms is the major route of ADMA clearance from the circulation, changes in DDAH expression levels and / or activity are a possible mechanism leading to ADMA accumulation and disturbance of endothelial homeostasis.

There is also plausibility of stereo-specific transporters of ADMA and SDMA. It is possible that alternative metabolic paths and cytological transporters are activated when renal function is impaired. Those alternative paths of metabolism of ADMA and SDMA may be more effective for SDMA.  However, our hypothesis assumes that the physiological factor should be sought, and not the biochemical parameters, for the relationship / correlation of ADMA vis GFR and SDMA vis GFR, as the very definition of GFR allows this to pass.

5. Did not mention about limitation of this study.

Answer : we added paragraph about limitations

Considering the overall design of our observation, it should be emphasized that our observations have limitations. It would be necessary to analyze more patients in each stage of CKD. Increasing  the numbers of children could sharpen the results towards statistically significant differences. It would also be interesting to expand the research group to include children after kidney transplantation. Another limitation of the study is the age characteristics of the patients, as the literature data show different values of ADMA concentration depending on age for the pediatric population [45]. Pharmacotherapy is also important, some drugs can modify ADMA levels. Therefore, the solution to these doubts, and at the same time preserving the benefits of the study group (e.g. lack of comorbidities, multi-drug pharmacotherapy, etc.), would be to deepen the research in the group of patients after adolescence, i.e. adolescent adults.

Reviewer 3 Report

In the manuscript entitled "Nitric oxide synthesis metabolites - as potential markers in chronic kidney disease in children" Piechowicz Joanna et al. aim to provide data regarding the biomarker potential of specific metabolites in the context of chronic kidney disease in children. For this purpose, the authors have organized a targeted study and aim to support their hypothesis through both between-groups and correlation analyses. Despite reducing the cohort number of each group, this grouping of subjects by their disease grading is really interesting and may provide novel information for the examined metabolites.

This Reviewer has some comments, as follow:

1.       Please provide the definition of GFR the first time you use it in the manuscript.

2.       Table 1 represents the differences in baseline biochemical characteristics between control and patient groups. Thus, it would be useful to be transferred in the Results section.

3.       Figure 3: It would be easier for the reader to follow if the authors could add marks of significance (where applicable) in the box-plots.

4.       Figure 4: The graphical presentation of the correlation analysis is very nice. Since the significance of the results is based on Spearman correlations, some of them are not easy to follow. Could you please indicate in the graph too (apart from the Table) which ones are significant and which not so that the reader doesn’t have to go back and forth? In addition, could you please add units of measurement in the axes?

5.       Discussion section: My main concern is that the authors do not focus on their novel findings, especially in the terms of disease grading. Some differences found between control groups and specific disease groups can be very useful towards the definition of easy to access biomarkers in early disease stages. To their credit, the authors have referenced the related bibliography, but I think that pointing out their novel findings and support them even through hypotheses would strengthen the impact of their findings.

6.       Conclusion section: This section resembles more a key-point list than a general conclusion of the findings. It would be more adequate to be presented as an overall concluding section including future research possibilities regarding the biomarker potential of NO-related metabolites.

Author Response

Dear Reviewer,

Thank you for your apt remarks giving us the opportunity to improve our manuscript.

1. Please provide the definition of GFR the first time you use it in the manuscript.

Detailed formula for estimation was added.

Answer : Taking GFR (glomerular filtration rate) values into account (estimated on the basis of Schwartz formula: eGFR (mL/min per 1.73 m²) = 0.413 ^* [height (cm)/serum creatinine (mg/dl)]), groups corresponding to a given stage of disease progression have been distinguished among CKD patients

2. Table 1 represents the differences in baseline biochemical characteristics between control and patient groups. Thus, it would be useful to be transferred in the Results section.

Answer : It was transferred to results section.

3. Figure 3: It would be easier for the reader to follow if the authors could add marks of significance (where applicable) in the box-plots.

Answer : marks were added.

4. Figure 4: The graphical presentation of the correlation analysis is very nice. Since the significance of the results is based on Spearman correlations, some of them are not easy to follow. Could you please indicate in the graph too (apart from the Table) which ones are significant and which not so that the reader doesn’t have to go back and forth? In addition, could you please add units of measurement in the axes?

Answer : We are really grateful for your appreciation. We merged table 4 and figure 4 to make it easier to read. We se your remark about adding units of measurement as valid however we decided not to include them as it would force us to make font smaller and thus less ridable. We consequently use the same units throughout whole article.

5. Discussion section: My main concern is that the authors do not focus on their novel findings, especially in the terms of disease grading. Some differences found between control groups and specific disease groups can be very useful towards the definition of easy to access biomarkers in early disease stages. To their credit, the authors have referenced the related bibliography, but I think that pointing out their novel findings and support them even through hypotheses would strengthen the impact of their findings.

Answer : Fragments discussing potential disease grading were added.

6. Conclusion section: This section resembles more a key-point list than a general conclusion of the findings. It would be more adequate to be presented as an overall concluding section including future research possibilities regarding the biomarker potential of NO-related metabolites.

Answer : We restructured conclusion section:

1. Children with CKD develop disturbances in most metabolites of NO synthesis.
2. These disorders worsen with the progression of CKD
3. Dialysis children treated show the greatest disturbances  of plasma ADMA and citrulline levels.
4. ADMA seems to be a good indicator of the gradual progression of the CKD, which is proved by the negative correlation with eGFR.
5. NO metabolites can possibly be promising markers of CKD stage and severity. Future research is needed to establish their clinical potential.

Round 2

Reviewer 1 Report

The authors improved the manuscript and responded to most of the comments appropriately. However, there are still some points that have to be clarified.

(1)    Abstract: Lines 20-22: It is stated that “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between groups within different stages of CKD (p ˂0.001).” However, Table 3 shows a p-value of 0.001 only for ADMA.

Answer: Sentence was corrected to “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between control vis CKD groups ( p<0.001-p<0.029). “

-          In the text of the revised version (lines 197 – 200) the significance values between the RRT group and the other groups are shown for ADMA and citrulline. However, in the abstract (lines 22 – 23) there is still stated that there were significant differences in the levels of SDMA and DMA as well.

-          Where does the value 0.029 comes from? What does it refer to?

(2)    It should be explained/commented why N is smaller than 48 or 33 for some parameters, and especially why N is 34 for Hb, Ht and RBC in the control group.

Answer: Data used in study is based on patients medical documentation. Sadly it was not always complete. Lack of data in some parameters is a result of incomplete patient documentation.

-          It is understandable that medical documentation is unfortunately not always complete. However, this does not explain the higher number (34) for Hb, Ht and RBC compared to the number of healthy children (33) given in the Methods section.

(3)    Table 2: N should be shown separately for the study and control group.

Answer: N was added

-          47 for the CKD group and 32 for the control group does not add up to 81, the number given in the heading! These inconsistencies do not contribute to the credibility of the data shown.

Author Response

Dear Reviewer,

Thank you for great remarks and carefulness during analyzing our work.  

(1)Abstract: Lines 20-22: It is stated that “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between groups within different stages of CKD (p ˂0.001).” However, Table 3 shows a p-value of 0.001 only for ADMA.

Answer: Sentence was corrected to “There were significant differences observed in levels of ADMA, SDMA, DMA and citrulline between control vis CKD groups ( p<0.001-p<0.029). “

-n the text of the revised version (lines 197 – 200) the significance values between the RRT group and the other groups are shown for ADMA and citrulline. However, in the abstract (lines 22 – 23) there is still stated that there were significant differences in the levels of SDMA and DMA as well.

Answer: Fragment (lines 22 – 23) in the new abstract is referring not to comparison between RRT group vs other groups but whole CKD children population vs healthy population. Those comparisons are summarized in table 2 (lines 191 – 193) and in results paragraph 3.1 (lines 185-189). Fragment was rephrased for clarity.

-Where does the value 0.029 comes from? What does it refer to?

Answer: This value comes from table 2 (lines 191 - 193)

(2)It should be explained/commented why N is smaller than 48 or 33 for some parameters, and especially why N is 34 for Hb, Ht and RBC in the control group.

Answer: Data used in study is based on patients medical documentation. Sadly it was not always complete. Lack of data in some parameters is a result of incomplete patient documentation.

-It is understandable that medical documentation is unfortunately not always complete. However, this does not explain the higher number (34) for Hb, Ht and RBC compared to the number of healthy children (33) given in the Methods section.

That was a mistake. It was corrected to 33.

(3)Table 2: N should be shown separately for the study and control group.

Answer: N was added

-47 for the CKD group and 32 for the control group does not add up to 81, the number given in the heading! These inconsistencies do not contribute to the credibility of the data shown.

We changed 81 to the correct number (79) and added information that data of 2 patients was missing.

Reviewer 2 Report

 Some data in supplement is wrong and no CKD stage 5 (non-dialysis) data.

Author Response

Dear Reviewer,

Thank you for your carefulness in anaylzing our manuscript.

Some data in supplement is wrong and no CKD stage 5 (non-dialysis) data.

Answer: In population of our study all stage 5 patients were on RRT. Detailed description of subgroups is in materials and methods section.

Mistakes in values of Hb were corrected.

Reviewer 3 Report

I have no other comments. The authors have replied my previous concerns.

Author Response

Dear Reviewer,

Once again thank you for your excellent remarks and the opportunity to improve our manuscript.

Round 3

Reviewer 2 Report

No questions.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.