Sex Differences in Fatty Acid Metabolism and Blood Pressure Response to Dietary Salt in Humans

Round 1

Reviewer 1 Report

This is an interesting and well done paper about an issue of general interest.

While I'm perfectly fine with the general conclusions here presented, I have a basic concern: the authors claim to evaluate salt sensitvity while they actually perform a difference analysis between low sodium and high (and not so high) sodium diets.

Sodium sensitivity is a subtle clinical entity (and a similarly subtle definition) which involves the study of BP variations (i.e. an increase of BP on a high- and a decrease of BP on a low-sodium diet), so quite different vs. the present study protocol where patients have been studied without intervention.

I believe the title and the discussion both should be changed consequently before a re-evaluation for a possible publication.

Author Response

Rev #1 Comments and Suggestions for Authors

This is an interesting and well-done paper about an issue of general interest.

1. While I'm perfectly fine with the general conclusions here presented, I have a basic concern: the authors claim to evaluate salt sensitvity while they actually perform a difference analysis between low sodium and high (and not so high) sodium diets.

We agree with the reviewer, we have changed this throughout the text. We have replaced describing our results from a salt sensitivity context and acknowledged it as blood pressure response to high salt and associated changes in metabolites in men and women. We discuss the implications of our findings to the trait of salt sensitivity and as the future direction to this study.

2. Sodium sensitivity is a subtle clinical entity (and a similarly subtle definition) which involves the study of BP variations (i.e. an increase of BP on a high- and a decrease of BP on a low-sodium diet), so quite different vs. the present study protocol where patients have been studied without intervention.

We agree that this study is directly assessing salt sensitivity. We have revised the manuscript to clarify this.

3. I believe the title and the discussion both should be changed consequently before a re-evaluation for a possible publication.

We thank the reviewer for this feedback. We have changed the title and discussion to better fit the experimental design and findings presented here.

Reviewer 2 Report

The present paper aimed to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake, revealing that excess Na+ is associated with fatty acid metabolism dysregulation and that the arachidonic acid signaling pathway may play a role in sex differences in salt sensitivity of blood pressure.  

A few changes are needed, as follows:

Materials and methods: Exclusion criteria are missing. Please provide!

Results, lines 125-126: “Evidence from clinical trials suggests that women have higher responsiveness of blood pressure to salt intake regardless of age and hypertension status.” Please remove this statement from Results and include it into Discussion.

3.1.1. Blood pressure and dietary Na+ intake in men and women: Please mention all variables which were significantly different in men and women according to Table 1.

Lines 138-139: You state:” Blood pressure response to dietary Na+ intake was higher in women than men.” Please provide some evidence for this statement!

Discussion: Please also mention that the variability in the ALOX5 gene was associated with carotid intima-media thickness, a marker of carotid atherosclerosis (Merlo et al, Arachidonate 5-lipoxygenase (ALOX5) gene polymorphism (rs12762303) and arachidonate 5-lipoxygenase activating protein (ALOX5AP) gene polymorphism (rs3802278) and markers of carotid
atherosclerosis in patients with type 2 diabetes mellitus. Int J Clin Exp Med 2016;9(2):4509-4514).

Author Response

Rev #2 Comments and Suggestions for Authors

The present paper aimed to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake, revealing that excess Na+ is associated with fatty acid metabolism dysregulation and that the arachidonic acid signaling pathway may play a role in sex differences in salt sensitivity of blood pressure. 

A few changes are needed, as follows:

1. Materials and methods: Exclusion criteria are missing. Please provide!

We thank the reviewer for bringing this crucial detail to our attention. We have added the exclusion criteria in the revised manuscript.

2. Results, lines 125-126: “Evidence from clinical trials suggests that women have higher responsiveness of blood pressure to salt intake regardless of age and hypertension status.” Please remove this statement from Results and include it into Discussion.

We have made this change.

3. 1.1. Blood pressure and dietary Na+ intake in men and women: Please mention all variables which were significantly different in men and women according to Table 1.

We thank the reviewer for this suggestion. This information has been added to the results section of the manuscript.

4. Lines 138-139: You state:” Blood pressure response to dietary Na+ intake was higher in women than men.” Please provide some evidence for this statement!

This statement was describing Figures 1B, 1C, and 1D specifically referring to the correlation analysis. The correlation looks more pronounced in women, we compared the slopes and found no statistical significance and have thus removed this statement from the discussion.

5. Discussion: Please also mention that the variability in the ALOX5 gene was associated with carotid intima-media thickness, a marker of carotid atherosclerosis (Merlo et al, Arachidonate 5-lipoxygenase (ALOX5) gene polymorphism (rs12762303) and arachidonate 5-lipoxygenase activating protein (ALOX5AP) gene polymorphism (rs3802278) and markers of carotid atherosclerosis in patients with type 2 diabetes mellitus. Int J Clin Exp Med 2016;9(2):4509-4514).

We thank the reviewer for this suggestion and have expanded the discussion to include this information.

Reviewer 3 Report

The study is an original contribution aimed to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake.  The manuscript presents a solid and very important study, focused on an important topic regarding the differences between genders in the context of sodium-dependent hypertension.

However, some issues must be resolved before the final approval of this study for publication.  The major issues are listed below:

Major:

Lines 53 – 57 – Here, the major fundament of this study seems confusing.  Is inflammation, in the classical point of view and with its five cardinal signals - rubor (redness), calor (warmth), tumor (swelling), dolor (pain), and functio laesa (loss of function) – the real starter of hypertension or are the inflammatory signaling, taking place under certain conditions, the real player?  More, is a change in the microbiome really producing systemic inflammation, considering the five cardinal signals above mentioned?  If so, hypertension could be resolved with anti-inflammatory therapy?

Lines 64 – 69 – Were the participants of the study hypertensive?  Also, considering the salt-sensitivity of blood pressure, established by Weinberger (1996), how salt sensitivity of blood pressure was tested in the subjects of this study.  If salt sensitivity of blood pressure was not tested, how differentiate salt-resistant from salt-sensitive subjects?

Lines 72 – 74 – As stated in the author’s discussion section, sodium consumption can be more precisely estimated using urine 24h-sodium excretion as an index.  Why did the author choose the use sodium intake in the food?

Table 1 – Were Eutrophic and overweight subjects compared within the same cohort?  If not, it would improve data interpretation since overweight is associated with hypertension (doi: 10.3892/etm.2016.3667).

Lines 255 – 257 – Are the man and women used for waist circumference at the same age and with similar body mass index?

Lines 269 – 292: The discussion in this paragraph seems quite speculative.  I would suggest an extensive review of this paragraph and a more concise approach.

Lines 314 – 320: Although very interesting, the evaluation of the gene expression in monocytes exposed to high sodium seems a little bit off in this study.  Since overall extracellular sodium concentration is kept within very narrow limits by many redundant controlling mechanisms, the physiological meaning of exposing circulating monocytes to high extracellular sodium concentrations looks out of context here.  Perhaps, it would be more concise and physiologically meaningful to evaluate such effects on immune cells of the small intestine, which would be more susceptible to high sodium gradients during the absorption processes.  How do the authors really interpret these findings in a physiological context?

Minor:

Line 31 - Please, check the units of the amount of sodium intake recommended by the AHA.  It seems oddly low.

Line 275 – Renal clearance of which substance?

Author Response

Rev #3 Comments and Suggestions for Authors

The study is an original contribution aimed to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake.  The manuscript presents a solid and very important study, focused on an important topic regarding the differences between genders in the context of sodium-dependent hypertension.

However, some issues must be resolved before the final approval of this study for publication.  The major issues are listed below:

Major:

1. Lines 53 – 57 – Here, the major fundament of this study seems confusing. Is inflammation, in the classical point of view and with its five cardinal signals - rubor (redness), calor (warmth), tumor (swelling), dolor (pain), and functio laesa (loss of function) – the real starter of hypertension or are the inflammatory signaling, taking place under certain conditions, the real player?  More, is a change in the microbiome really producing systemic inflammation, considering the five cardinal signals above mentioned?  If so, hypertension could be resolved with anti-inflammatory therapy?

We thank the reviewer for these important questions. Evidence supports that chronic low-grade inflammatory signaling is the key player to the pathogenesis of diseases like hypertension. At this time, there is not enough evidence to prove whether the system inflammation is directly produced by the microbiome or vice versa. Studies show that this is a bidirectional relationship and in some diseases such as cancer, the five cardinals mentioned above maybe present. The five cardinals are less prevalent in hypertension which is why the disease is also referred as a silent killer unless accompanied with comorbidities that exhibit the symptoms. The resolution of hypertension with anti-inflammatory therapy remains a topic of research. In the recent CANTOS trial (Canakinumab Anti-inflammatory Thrombosis Outcome Study), inhibition of IL (interleukin)-1β using the human monoclonal antibody canakinumab reduced inflammatory markers but did not reduce development of incident hypertension (PMID: 30586730).

2. Lines 64 – 69 – Were the participants of the study hypertensive? Also, considering the salt-sensitivity of blood pressure, established by Weinberger (1996), how salt sensitivity of blood pressure was tested in the subjects of this study.  If salt sensitivity of blood pressure was not tested, how differentiate salt-resistant from salt-sensitive subjects?

We thank the reviewer for these questions. The key characteristic of salt sensitivity is changes in dietary sodium intake that mirror changes in blood pressure. Accordingly, we used the relationship between dietary salt intake and blood pressure in these participants. We acknowledge that this is not a rigorous measurement and hence why we refer to the analysis as a blood pressure response to salt intake instead of salt sensitivity. We are currently recruiting participants to repeat this study in hypertensive patients who undergo the Weinberger protocol to diagnose salt sensitivity. We have revised the manuscript and title to reflect that this is not a direct study of salt sensitivity.

3. Lines 72 – 74 – As stated in the author’s discussion section, sodium consumption can be more precisely estimated using urine 24h-sodium excretion as an index. Why did the author choose the use sodium intake in the food?

We agree with the review that a metric such as the 24h sodium urinary excretion would have been a far better tool for this purpose as acknowledged in our discussion. We chose to use sodium intake in the food because this study is cross-sectional by design. We are currently recruiting participants to repeat this study in hypertensive patients who undergo the Weinberger protocol to diagnose salt sensitivity. The patients are admitted into the clinic for 3 days and we collect blood and 24h urine to have use various gold standard methods to assess salt-sensitivity.

4. Table 1 – Were Eutrophic and overweight subjects compared within the same cohort? If not, it would improve data interpretation since overweight is associated with hypertension (doi: 10.3892/etm.2016.3667).

We agree with the reviewer that there is an association between obesity and hypertension as supported by numerous lines of evidence. All subjects enrolled in this study were compared within the same cohort irrespective of their weight status. The goal of this study was to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na⁺ intake. We assessed metabolites of 44 women and 31 men. We refrained from comparing subjects based on weight due to a limited sample size within the context of sex differences as the analyses would have been statistically underpowered.

5. Lines 255 – 257 – Are the men and women used for waist circumference at the same age and with similar body mass index?

We thank the reviewer for this question. We found that among the subjects in this study, women were younger than men (28.68 ± 7.953    vs. 32.71± 9.32, P=0.0481). Men had a higher body mass index 27.14 ± 5.57 vs. 24.27 ± 6.17, P=0.0423) and larger waist circumference (91.69 ± 15.18 vs. 76.76 ± 13.37, P<0.0001). We have included this information in Table 1 and revised the manuscript to include this information.

6. Lines 269 – 292: The discussion in this paragraph seems quite speculative. I would suggest an extensive review of this paragraph and a more concise approach.

We thank the reviewer for this feedback. This paragraph discusses metabolomic sex differences found in men and women within and outside of the dietary salt context. This is an avenue of research that is yet to be explored. Here, we wanted to discuss our results but also shed a light on potential mechanistic links with hypertension based on the limited available evidence on the metabolites. We are currently using preclinical models to investigate these pathways.

7. Lines 314 – 320: Although very interesting, the evaluation of the gene expression in monocytes exposed to high sodium seems a little bit off in this study. Since overall extracellular sodium concentration is kept within very narrow limits by many redundant controlling mechanisms, the physiological meaning of exposing circulating monocytes to high extracellular sodium concentrations looks out of context here.  Perhaps, it would be more concise and physiologically meaningful to evaluate such effects on immune cells of the small intestine, which would be more susceptible to high sodium gradients during the absorption processes.  How do the authors really interpret these findings in a physiological context?

The lack of differences in renal handling of a salt load or plasma volume between salt-sensitive and salt-resistant individuals suggests extrarenal mechanisms which certainly includes the gastrointestinal tract. In fact, our recent publication that explored the effects of salt on the gut microbiome in these participants provides evidence of high salt on immune cells of the intestine. We reported that colon sections of hypertensive humans had higher T cells and macrophages infiltration than normotensive. We further showed these findings in mice. However, we also showed this inflammatory phenotype extends to other organs including the spleen. Salt sensitivity is a devastating disease, and we are interested in identifying novel therapeutic approaches. Evidence supports that immune cells such as monocytes can sense Na⁺ contribute to the homeostasis. We recognize that experimentally evaluating immune cells in intestines of healthy living human participants would be extremely challenging. Additionally, our group and others have shown that adoptive transfer of immune cells derived from organs such as the spleen affects the hypertensive phenotype (PMID: 35862128, PMID: 26595812) cementing that immune cells play a big role even when they are not from the intestines directly. We believe there is great translational relevance in using human monocytes. Collectively, we believe these provide a strong rationale to evaluate the gene expression in monocytes exposed to high sodium since we already showed effects on immune cells of the small intestine in high salt and hypertension.

Minor:

8. Line 31 - Please, check the units of the amount of sodium intake recommended by the AHA. It seems oddly low.

We have double checked that the recommendation by AHA is 2.3 g of Na⁺ or 5 g of salt per day. (https://www.heart.org/en/healthy-living/healthy-eating/eat-smart/sodium/how-much-sodium-should-i-eat-per-day).  Other international organizations including WHO recommend similar limits (https://www.who.int/news-room/fact-sheets/detail/salt-reduction).

9. Line 275 – Renal clearance of which substance?

We apologize for the confusion here. We have revisited this line to clarify that hippurate clearance is used to assess effective renal plasma flow.

Round 2

Reviewer 1 Report

I appreciated the revision made by the authors.

I believe the title should be changed accordingly before a possible evaluation for publication.

Author Response

Rev 1 - Comments and Suggestions for Authors

I appreciated the revision made by the authors.

Thank you

I believe the title should be changed accordingly before a possible evaluation for publication.

We thank the reviewer for reminding us of this crucial detail. We have changed the title accordingly.

Reviewer 3 Report

The authors provided important enlightenment on several questions raised in the previous version.  However, the manuscript by itself seems to have very few benefits from these enlightenments.

1 – The mention of salt-sensitivity of blood pressure should be reconsidered in the title.

2 – The introduction section should include a more consistent presentation of the problem, in light of the low-grade inflammation, its difference from the classical inflammation concept, and its correlation with blood pressure and salt consumption.

3 – Regarding the monocytes gene expression experiment, although interesting, a contextualization is still missing.  For instance, the authors discussed (in the answer to the reviewer) the question raised in the previous version concerning the physiological meaning of exposing circulation monocytes to a high-sodium medium, but they did not include such a discussion in the manuscript, providing a rationale for the assay.  I acknowledge the difficulties in extracting inflammatory cells from human subjects, but a contextualization of the results based on recent findings would greatly improve the discussion section. 

Author Response

Rev 3 - Comments and Suggestions for Authors

The authors provided important enlightenment on several questions raised in the previous version.  However, the manuscript by itself seems to have very few benefits from these enlightenments.

1 – The mention of salt-sensitivity of blood pressure should be reconsidered in the title.

We thank the reviewer for this comment and have revised the title to remove salt-sensitivity.

2 – The introduction section should include a more consistent presentation of the problem, in light of the low-grade inflammation, its difference from the classical inflammation concept, and its correlation with blood pressure and salt consumption.

We have expanded the introduction to include more details about inflammation, its correlation with blood pressure and salt consumption.

3 – Regarding the monocytes gene expression experiment, although interesting, a contextualization is still missing.  For instance, the authors discussed (in the answer to the reviewer) the question raised in the previous version concerning the physiological meaning of exposing circulation monocytes to a high-sodium medium, but they did not include such a discussion in the manuscript, providing a rationale for the assay.  I acknowledge the difficulties in extracting inflammatory cells from human subjects, but a contextualization of the results based on recent findings would greatly improve the discussion section. 

We agree with the reviewer and have revised the discussion accordingly. In brief, we contextualize this section by highlighting that previous research by our group and others have identified monocytes as key antigen presenters that contribute to blood pressure regulation in humans. While we cannot conduct this experiment in humans, we infer that using this previously validated in vitro assay in human monocytes treated with high salt is a step in the direction of identifying specific mechanisms by which fatty acid metabolism may be linked to high salt and inflammation in hypertension and salt sensitivity. We hope to explore these mechanisms in mice models of high salt-induced hypertension and inflammation.