Differential Effects of Alcoholic and Non-Alcoholic Beer Intake on Renin–Angiotensin System Modulation in Spontaneous Hypertensive Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for permitting me to review this manuscript 

In this study the authors assessed the effect of  beer on different types of rats , they concluded that  on RAS enzyme activity  and other factors 

They conclude that Significant changes occurred in RAS enzyme activity across tissues,  Moderate beer consumption did not elevate blood pressure  Nevertheless, beer modulated RAS enzyme activity, indicating potential impacts 22 on cardiovascular homeostasis

I have some suggestions 

the chapter on the results concerning modulation of RAS activity may need some clarification , at least a summary  helping the reader to retain a concise message in addition the authors may immagine possible  future clinical implications 

please elaborate the fact the modulation of tha RAS did not affect BP and pulse rate 

 

Comments on the Quality of English Language

english is fine 

Author Response

Thank you for your valuable feedback. We appreciate your comments and suggestions. Therefore, we have made the following changes:

1. We have summarized the results related to RAS modulation to help the reader to retain the most important results.

To summarise the results concerning the modulation of the renin angiotensin system, significant changes of RAS enzyme activity have been found in several tissues, including the hypothalamic-pituitary-adrenal (HPA) axis, the kidneys and the lungs. Changes in the HPA axis indicate that beer consumption may influence neuroendocrine regulation. Changes in the kidney suggest that beer consumption may affect renal mechanisms involved in long-term blood pressure control. In the lungs, changes in angiotensin-converting enzyme (ACE) activity suggest that beer may influence the conversion of angiotensin I to angiotensin II.

2. We have added a paragraph with potential clinical implications.

Some future clinical implications that could be explored are to analyse the effects of beer consumption in individuals with a genetic predisposition to hypertension.  Given that significant differences in the response to beer consumption occur between WKY and SHR rats, it is possible to investigate whether beer consumption, even in moderate amounts, might have a different impact on individuals with a genetic predisposition to hypertension. The impact of beer consumption on long-term kidney function may also be considered. Longitudinal studies in humans could assess whether regular beer consumption is associated with changes in kidney function or an increased risk of chronic kidney disease.

3. We have tried to explain why blood pressure and pulse rate are not affected by RAS modulation.

We have showed that beer consumption does not modify blood pressure levels or heart rate. Thus, both blood pressure and heart rate levels are not decreased in hypertensive animals, nor are they increased in normotensive animals. Several factors can be considered to explain this phenomenon, such as the presence of compensatory mechanisms or differences between systemic and local regulation of RAS. The circulatory system has an extensive mechanism to control blood pressure. If the pressure deviates from its normal mean value, nerve reflexes are activated within seconds that trigger changes to restore the pressure to normal levels. This may include adjustments in ex-tracellular fluid volume, and thus blood volume, to increase or decrease blood pres-sure. The observed changes in RAS regulatory enzyme activities, which vary according to the type of beer ingested and the group of animals, could be the result of these compensatory mechanisms acting to maintain stable blood pressure. Individual varia-bility and genetic predisposition must also be considered: WKY and SHR rats showed distinct patterns of RAS modulation in response to beer consumption. This suggests that genetic predisposition, such as the hypertensive phenotype in SHR rats, plays a role in determining how beer consumption influences regulatory systems such as RAS. It is plausible that the lack of significant changes in blood pressure and heart rate is due, in part, to the resistance to developing hypertension in WKY rats, even in the face of RAS modulation. Finally, we should not lose sight of the possible long-term effects and that the effects of beer consumption on blood pressure and heart rate may be manifested in the longer term. Additionally, the occasional modifications found could be due to phenomena related to animal handling, as they are not changes that evolved throughout the study period. Therefore, beer seems to exert a positive effect on cardiovascular disease [25-34,48-55] and our results support that beer, alcoholic or non-alcoholic, at least not produce negative effects on the development of hypertension.

I would like to express my sincere gratitude for the invaluable comments you have provided, which have significantly enhanced the quality of our manuscript. Your insights and feedback are truly appreciated.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the effects of alcoholic (AB) and non-alcoholic beer (NAB) consumption on blood pressure and the activity of enzymes regulating the renin-angiotensin system 10 (RAS) in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).

The idea of this study - is principally interesting and this manuscript is well-written.  However, this manuscript needs some improvements and corrections before publishing may be possible.

 

Keyword: please add also to: rats.

For better understanding for reader please add a time-line of all your experiments as a Figure 1.

 

Introduction

Lines 28-30: please add multiple references at the end of each these sentences.  

Please add also a hypothesis to your Introduction section, which will be rejected or confirmed in the Discussion section.  

 

Materials and Methods

First of all, please add also date for permission of all these experiments.

Please add he references for each single method used in this study.

Lines 172-176: please add the exactly description of the anesthesia of the animals.

 

Discussion

Please discuss all your results deeper and please add also more explanation of your results.

 

Conclusions

This conclusion is too long, please draw a more concrete Conclusion and please add the mostly information to the Discussion section.

 

Please add also a Limitation section to your manuscript.

Author Response

Thank you very much for your review of our manuscript. According to your suggestions, we have made the next changes:

1. “Rats” has been added to keywords.
2. A diagram with the chronology of the experiments has been included.
3. References have been included.
4. Hypothesis has been included and discussed.
5. Please, note that the code number of the IACUC of UJA includes the date for experimental permission.
6. References of methods have been included.
7. Please, note that anesthesia has been previously described in section 2.3.
8. Several changes have been done along the manuscript, also according to other reviewers’ comments.
9. In response to your suggestion regarding the conclusions section, we have decided to maintain the conclusions as they were originally presented. Our rationale for this decision is that the current conclusions effectively summarize the diverse and extensive set of results presented in the study. Given the complexity and variability of our findings, we believe that the conclusions, as they stand, provide a comprehensive overview that ties together the key points of our research.
10. A section of limitations has been included.

I would like to express my sincere gratitude for the invaluable comments you have provided, which have significantly enhanced the quality of our manuscript. Your insights and feedback are truly appreciated.

Reviewer 3 Report

Comments and Suggestions for Authors

The present study aimed to assess the effects of consumption of alcoholic beer (AB) and non-alcoholic beer (NAB) on various cardiovascular parameters in healthy Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR), particularly focusing on investigation of the involvement of RAS-regulating enzymes in blood pressure control. The study is generally well designed, the methods are appropriate, and the obtained results provide valuable insights into the complex interactions between beer consumption, RAS modulation, and cardiovascular health. There are, however, some concerns, both essential and technical, which need to be addressed.

Firstly, the manuscript is too long and difficult to follow. It needs to be reorganized; parts of the manuscript should be summarized in supplementary figures.

Secondly, parts of the text cannot be read since the figures cover them (pages 6 and 7). Besides, some figures are not in the proper place in the manuscript (e.g., Figure 1 is in Methods). There are also two figures 5 in the manuscript.

One of the major concerns is the characterization of the beer used. Beer composition can vary significantly depending on its style, ingredients, and brewing process. All these details have to be clear in the text.

The purpose of the animal experimental groups with animals choosing tap water or beer (TW/NAB and TW/AB) is not clear in the text, and the significance of the differences between these groups and NAB and AB groups is not thoroughly discussed in the manuscript.

How was “moderate” and “non-moderate” beer intake defined in this study?

The abstract needs to be rewritten: (1) it needs to be clear in the abstract that WKY rats were actually normotensive controls for SHR rats; (2) the term “RAS enzyme activity” is incorrect and should be rephrased, as it is in the main text.

Line 86: “beer has always been part of the classic Mediterranean diet” – I disagree with this statement; beer began to gain popularity in Mediterranean countries in more recent centuries, especially with the influence of northern European beer cultures. The Mediterranean region has long been associated with wine production and consumption, dating back thousands of years.

Some terms, such as “perfectly compatible” (line 94) represent overstatements that should be rephrased in my opinion. There are similar examples in the text.

Based on the above, I suggest the substantial reorganization of the manuscript with solving the technical issues before resubmitting it.

Comments on the Quality of English Language

I suggest additional language editing. Some sentences represent overstatements that need to be rephrased.

Author Response

1. We sincerely appreciate the reviewer's diligent assessment of our manuscript. While we acknowledge the substantial length of the article, we respectfully contend that the comprehensive information presented therein is of paramount importance to the field. In an effort to enhance readability and facilitate a more structured comprehension of the material, we have meticulously organized the content into distinct sections. This deliberate segmentation is intended to provide readers with a clear navigational framework, enabling them to efficiently parse the information and, if desired, focus on specific areas of interest. We believe that this approach not only maintains the integrity and depth of the research but also optimizes the reader's ability to engage with and assimilate the complex data and analyses presented. We are, of course, open to any suggestions for further refinement that the reviewer may deem beneficial.
2. We deeply regret the unfortunate formatting issue that has arisen due to the inclusion of figures in the Word document, which has inadvertently altered the structure of the text and rendered certain sentences illegible. We will try to correct it without changing the layout of the journal.
3. We have described the beer used in our experiments.
4. We have included the rationale to include groups choosing tap water or beer: Experimental groups where rats could choose between tap water (TW) or beer (either alcoholic, AB, or non-alcoholic, NAB) were included in the study to investigate how voluntary preference for beer might affect the parameters studied. In other words, these groups seek to determine whether the animal's free choice influences consumption, body weight, blood pressure and RAS enzyme activity compared to groups where only one type of beverage was provided.
5. We have included a paragraph regarding the concepts of moderate and non-moderate intake. The research includes groups where the rats had unlimited access to beer (AB or NAB). This suggests that intake in these groups could be considered ‘unmoderated’, as the animals could consume as much as they wished. The inclusion of groups where rats could choose between tap water (TW) and beer (TW/AB and TW/NAB) allows exploration of a range of intake that could be considered ‘more moderate’ than that of the groups with unrestricted access. In any case, it is true that recommendations on moderate alcohol consumption in humans are not directly applicable to animal studies.
6. The proposed corrections of the summary have been included.
7. We acknowledge and appreciate the reviewer's observation regarding the historical context of beer in the Mediterranean diet. Indeed, we concur that wine has traditionally been the more classical beverage associated with this dietary pattern over the centuries. However, we have considered a more contemporary timeframe. In this recent historical context, beer has indeed become an integral part of the modern Mediterranean dietary landscape. In any case, we have simplified the phrase to avoid confusion.
8. Some expressions have been rewritten according to the reviewer’s comments.

I would like to express my sincere gratitude for the invaluable comments you have provided, which have significantly enhanced the quality of our manuscript. Your insights and feedback are truly appreciated.

Reviewer 4 Report

Comments and Suggestions for Authors

Hypertension is a serious public health issue with a significant impact on the global burden of disease. It is one of the leading risk factors for mortality associated with cardiovascular diseases, including myocardial infarction and stroke. Its increasing prevalence poses a substantial challenge to healthcare systems worldwide. Undoubtedly, the study entitled “Differential Effects of Alcoholic and Non-Alcoholic Beer intake on Renin-Angiotensin System Modulation in Spontaneous Hypertensive Rats” has an applied aspect, as it aligns with addressing this issue.

The study is highly engaging and addresses the topic of hypertension in rats. It offers valuable insights into the biochemical mechanisms underlying the effects of alcoholic and non-alcoholic bear on hypertensive rats.

I did not find any methodological errors in the study. However, I have a few comments, which I outline below:

1.       Figure 1: I suggest presenting the data in a different format or improving the quality of the graphs.

2.       As is well known, hops reduce blood pressure and have diuretic effects. Could these results be linked to the observations made in the study? The authors should consider discussing the active substances that might be responsible for the observed effects.

3.       Section 2. Materials and Methods: It is well known that there are various types of beer. The authors should provide a characterization of the specific beer used in the study. This is not merely a question of distinguishing between alcoholic and non-alcoholic beer. An analysis of the natural substances contained in the tested beer would be beneficial to evaluate which components influence the observed effects. While alcohol itself undoubtedly plays a role, other ingredients, such as hops mentioned earlier with its diuretic properties, are also likely to contribute to the observed outcomes.

Author Response

Thank you very much for your comments. We have made the next changes:

Figure 1 has been redrawn for a much better observation.

We have included information about the putative effects of additional bioactive components of beer on the results. Certainly, beer contains a variety of bioactive components, such as B vitamins: folic acid, riboflavin, niacin, thiamine, pantothenic acid, biotin and vitamin B12; minerals: magnesium and calcium; soluble fibre, natural antioxidants and other substances derived from malted barley, hops, water and yeast. Although our study does not specifically analyse the impact of these bioactive components, some possible effects are antioxidant, anti-inflammatory effects, effects on lipid metabolism or interactions with the endocrine system. Antioxidants may neutralise free radicals, which are known to be involved in the development of hypertension; some components of beer, such as polyphenols, have anti-inflammatory properties. Inflammation also plays a role in the development of hypertension. B vitamins and soluble fibre present in beer may have contributed to the observed changes in lipid profile. Finally, some components of beer may interact with the endocrine system, including the RAS, through mechanisms not yet fully understood.

We have described the beer used.

I would like to express my sincere gratitude for the invaluable comments you have provided, which have significantly enhanced the quality of our manuscript. Your insights and feedback are truly appreciated.

 

Reviewer 5 Report

Comments and Suggestions for Authors

The relationship between alcoholic and non-alcoholic beverages and the renin-angiotensin system is well established. Alcohol intake initially suppresses the activity of this system by inhibiting the release of renin.

 

Your abstract should be structured but without headings. First, provide the background by placing the research question in a broad context and highlighting the study's purpose. Next, briefly describe the main methods or treatments used, including any relevant preregistration numbers and the species and strains of any animals involved. Then, summarize the main findings of the article. Finally, indicate the primary conclusions or interpretations drawn from the study. The abstract must objectively represent the article; it should not include results that are not presented and substantiated in the main text, and it should avoid exaggerating the conclusions. Consider adding some conclusions; the abstract does not indicate what this study contributes.As early as in 1928, you agree that "Blood pressure depends on the interaction of several factors." Further introduction is tedious and lengthy, but it is barely mentioning any.It is no surprise then that "alcoholic beer" and "nonalcoholic beer" remained obscured until lines 101 and 102. Likewise, the "introduction" section lacks a unanimous aim.I believe a sentence from line 31 is more appropriate as the lead sentence in the article.Ln 41 - Change "ejzymatic protein" to "enzyme."

 

 

Author Response

Thank you for your feedback.

According to your comments and the several reviewers’ comments, substantial changes have been made to the manuscript. I would like to express my sincere gratitude for your thorough and insightful review of our manuscript. Your detailed comments and suggestions have significantly contributed to improving the quality and clarity of our work. We appreciate the time and effort you have invested in providing such valuable feedback.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for all corrections. 

Author Response

Thank you very much for the time and effort you have put into reviewing our manuscript.

Kind regards

Reviewer 3 Report

Comments and Suggestions for Authors

The authors modified the initial version of their manuscript according to my suggestions. I still think that the manuscript structure should be reorganized, with parts of the results being in the supplementary material; however, I will support the publication in this form.

Author Response

Thank you very much for the time and effort you have put into reviewing our manuscript.

Kind regards

Reviewer 5 Report

Comments and Suggestions for Authors

Substandard

I am not convinced that the author's conclusions would change for other alcoholic beverages compared to their non-alcoholic counterparts. True objectivity can only be attained through a blind experiment (for the observeras well). Without this, the final conclusions may be influenced by the observer's personal biases.

 

Author Response

Thank you very much for the time and effort you have put into reviewing our manuscript.

Kind regards