Characterization of the Impacts of Living at High Altitude in Taif: Oxidative Stress Biomarker Alterations and Immunohistochemical Changes

Round 1

Reviewer 1 Report

"Screening impacts of living at high altitude in Taif area: Oxidative stress, Biomarkers alterations, and immunohistochemical changes" by Soliman and colleagues is an original paper investigating changes in the liver and kidney of rats after exposure to high altitude. The authors conducted histological, immunohistochemical and genetic analyses and found an increase in biomarkers of tissue damage and inflammatory cytokines in the high-altitude group compared to the control group at sea level. Thus, they suggest that the presence of tissue damage and the expression of biochemical and molecular markers may represent critical therapeutic targets for living at high altitude.

Although the work is interesting, it needs some improvements.

Introduction. I suggest that the authors rework the introduction, as the sentences often seem unrelated. Also, in lines 82-86, the authors should better define the purpose of the work, referring also to the experimental model used.

Materials and Methods.
- In section "2.1. Animal handling and experimental design", the authors should specify the strain of mice used. In addition, they should use the same subdivision into groups, since they referred to two groups, A and B, in the abstract. 
- Please check the division and order of the different paragraphs.

Results
- The quality of figures 3 and 4 is not good as the images are blurred. I suggest the authors replace these images with higher resolution ones to better observe the details described in the text.

Author Response

Reply to reviewer 1 comments (cimb-16465002)

Dear respected reviewer, great thanks for your valuable comments that strength our paper and we have revised the manuscript based on your comments.

The tracked file contain the reply for your comments in original submitted word file.

The paper has been edited for language using MDPI language editing service.

The certificate of language editing is attached.

There are 2 files of clean revised and edited paper.

Please see down our reply for your respected comments

"Screening impacts of living at high altitude in Taif area: Oxidative stress, Biomarkers alterations, and immunohistochemical changes" by Soliman and colleagues is an original paper investigating changes in the liver and kidney of rats after exposure to high altitude. The authors conducted histological, immunohistochemical and genetic analyses and found an increase in biomarkers of tissue damage and inflammatory cytokines in the high-altitude group compared to the control group at sea level. Thus, they suggest that the presence of tissue damage and the expression of biochemical and molecular markers may represent critical therapeutic targets for living at high altitude.

Although the work is interesting, it needs some improvements.

Introduction. I suggest that the authors rework the introduction, as the sentences often seem unrelated.

Authors reply:

The introduction was totally formatted and constructed.

Also, in lines 82-86, the authors should better define the purpose of the work, referring also to the experimental model used.

Authors reply:

Last part of introduction that contain the purpose of current study was directed as you suggested.

 

Materials and Methods.

- In section "2.1. Animal handling and experimental design", the authors should specify the strain of mice used. In addition, they should use the same subdivision into groups, since they referred to two groups, A and B, in the abstract. 
- Please check the division and order of the different paragraphs.

Authors reply: All your respected points have been considered in the revised file.

Results
- The quality of figures 3 and 4 is not good as the images are blurred. I suggest the authors replace these images with higher resolution ones to better observe the details described in the text.

Authors reply: Figure 3 and 4 was replaced by high resolution photos. New description for results and legends were explained in revised file.

Paper has been edited by MDPI editing language and certificate was attached.

 

Author Response File: Author Response.doc

Reviewer 2 Report

The manuscript presents the results of experimental work, the topic of which is relevant, timely and interesting for a wide range of readers. However, the presentation of the data and the style of presentation of the text are not yet ready for publication. The comments are set out below.

Title:  stress, Biomarkers - if you delete the comma here, it might be better

Lines 19-20: the meaning of the sentence is not clear

Line 21: it is necessary to determine which alterations in the liver and kidneys the authors are going to investigate

 Line 24:  blood was collected from the orbit - and according to Methods - blood was from the tail vein (line 93), so фuthors should clarify this information

 Lines 25-26: Liver and kidney tissues were taken for histological, immunohistochemical, and genetic changes - the text this sentence does not suggest at all what the authors undertook in their work

Line 27:   AST, ALT were not studied in this work

Lines 27-28: According to the text of this sentence, SOD, catalase and NO were studied in kidney and liver, but in the results section it is written that they were studied in blood serum. It is not clear why nothing is written about GSH

 Line 29:   marker was increased on groups - according to the content of the article, there was only one group, perhaps the authors wanted to write rats. And in general, throughout the text it is better to write "in rats from such and such a group", and not just in a group. Throughout the text, authors should more precisely and clearly articulate what they want to express.

Lines 34-36: The meaning of the final sentence in the abstract is not clear. The authors need the help of a professional translator.

Line 49: repetition of the abbreviation given on line 48

Lines 61-62, 81-86: English problems

The introduction does not clearly state the aims of the study.

Line 88: it is necessary to indicate how old the rats were and in what conditions (above sea level) they lived from birth to the beginning of the experiment

Line 93: English problems

 Line 101: reduced glutathione (GSH),- why is the word reduced  needed here?

Line 104: GPT, GOT - please explain the abbreviations

Line 105: using Spectrum Co. kits - it is necessary to give an exact description of the kits

 Line 114: what 260/280 ratios were obtained and considered acceptable for PCR?

 Lines 118-119 - text repetition

Line 120, 121, 123, 134, 156, 165: English problems

Line 120: According to the obtained cytological data, it can be assumed that the cell cytoskeleton changes during hypoxia. This may mean that beta-actin can change the level of expression and, if so, the authors cannot use its expression as a reference gene when performing real-time PCR.

Lines 129-130: shift text relative to the table

Table 1: you need to enter the names of the genes,

Line 138: hemoxugenase-1 - gross misprint

Line 140-143: HO-1 - The abbreviation was not entered, English problems, sentences are written very complicated and difficult to read

Lines 145-146: information (objective 40)/marker/organ/animal (40 HO-1 145 images and 40 Nrf2 images/organ/group) is hard to understand

Lines 147- 148: It is not clear what +Ve and -Ve are. Why is the ";" sign used after the word scale?

Tables 2 and 3, and 4, and 5, and Figures 1, and 2: the authors must explain what the letters a and b in the table mean

Line 174: “ per each experiment.” – you had only one experimental group,  did you mean “per each group”? There is not a single * sign indicating significant differences in the table

Line 177: why only MDA, catalase, and SOD are listed, but what about NO and GSH?

Line 181: same as for line 177

Line 189: when writing IFN gamma, sometimes a hyphen is used, and sometimes for some reason not. Same for other genes (EGLN-2, Nrf-2). Check the whole text, please

Table 3: correct the spelling of all symbols.

 Line 189: the abbreviation HA was not introduced

Line 190: inflmmations

Line 191: taif region. – probably Taif region.

Lines 204-211: the first sentence is very confusing. Please describe everything about Figure 1 first and then about Figure 2.

Line 207: Tere were

Line 210: “There was no changes in the VHL mRNA expression between both high altitude and normal sea level.” - Please read and think about what you have written. It turns out that you characterize the sea level by the level of gene expression. And this problem is all over the text. It is necessary to make the text understandable for the reader.

Lines 235-236 and 258-259: oxidative tress  quantitativereal - improve, please;

Lines 235-237, 258-260: Figures 1 and 2 show not Impacts of high altitude on hepatic genes, but Impacts of high altitude on hepatic genes expression; hepatic genes cannot be associated with biomarkers - please check the meaning of the sentence and the overall correctness of the translation. 10 different rats per treatment - the word treatment can only be used in relation to one group. The label of the Y axis is not true, it reflects the general content of the figure, but not what the numbers on the Y axis indicate.

Line 265: “hepatic tissue of high attitude group” - All phrases like this should be corrected in the text of the entire manuscript. For example, this particular sentence should be corrected to “hepatic tissue of  rats from high attitude group”

Lines 263-276: It is customary to refer to the figures in order - first 3a, then 3b, then 3c and then 3d. the same for figure 4/

Line 302: attitude

Line 304: stained liver of the high attitude group

Line 307: attitude group

Legend for Fig.4 also contains many mistakes

Line 338:” Lesion scoring as described by H& E“ - I guess H&E can't describe anything

Lines 346-351: many mistakes

 Line 352, 365: English problems

Line 418: why it is written IFN alfa?

Line 420: Why do you think there is only one pathway involved?

Line 421: English problems

Line 424:  high altitude groups - why do you write that there were several such groups in the experiment?

Line 500: in many rat organs - You analyzed only two organs

Line 506: “ Cautions must be taken, and more health care must be directed to people living at high altitudes.” - This phrase is not very correct, since the local population, permanently living at an altitude of 2500 m above sea level, is unlikely to have such consequences as described in the experiment. Rather, this applies to tourists who come to rest.

 

Make gene symbols throughout the text in italics

Reading the manuscript is difficult because the authors use numerous abbreviations, many of which have never been deciphered. There is also no list of abbreviations.

 

The text contains a significant number of typos and translation errors. This list of comments does not list all errors and typos.  Also, there are a lot of phrases in the text that incorrectly reflect the meaning of what the authors wanted to say. It is necessary to carefully check the content of the text and edit the manuscript with the help of a professional translator or native speaker.

 

Author Response

Reply to reviewer 2 comments

Dear respected reviewer, great thanks for your valuable comments that strength our paper and we have revised the manuscript based on your comments.

The tracked file contain the reply for your comments in original submitted word file.

The paper has been edited for language using MDPI language editing service.

The certificate of language editing is attached.

There are 2 files of clean revised and edited paper.

Please see down our reply for your respected comments

The manuscript presents the results of experimental work, the topic of which is relevant, timely and interesting for a wide range of readers. However, the presentation of the data and the style of presentation of the text are not yet ready for publication. The comments are set out below.

Title:  stress, Biomarkers - if you delete the comma here, it might be better

Authors reply:

You suggestion was done correctly.

Lines 19-20: the meaning of the sentence is not clear

Authors reply:

 The sentence was changed to be:

 At high elevations, the human body suffers a number of pathological, physiological, and biochemical changes, all of which have an adverse impact on human health and organs vitality.

Line 21: it is necessary to determine which alterations in the liver and kidneys the authors are going to investigate

Authors reply:

This sentence was changed to be:

This study aimed to investigate the alterations in the liver and kidney biomarkers, oxidative stress markers, gene expressions and cellular histology of rats maintained at high altitudes and normal sea levels.

 Line 24:  blood was collected from the orbit - and according to Methods - blood was from the tail vein (line 93), so authors should clarify this information

Authors reply:

The blood was collected from the orbit. We changed the written word in line 93

 Lines 25-26: Liver and kidney tissues were taken for histological, immunohistochemical, and genetic changes - the text this sentence does not suggest at all what the authors undertook in their work.

Authors reply: The sentence was reconstructed to be:

 Liver and kidney tissues from both groups were taken to examine the heptorenal changes at histological, immunohistochemical, and genetic levels.

Line 27:   AST, ALT were not studied in this work

Authors reply: GPT and GOT were inserted instead of AST and ALT as both are the same.

Lines 27-28: According to the text of this sentence, SOD, catalase and NO were studied in kidney and liver, but in the results section it is written that they were studied in blood serum. It is not clear why nothing is written about GSH.

Authors reply:

Serum word were added for the sentence and GSH was inserted with SOD, catalase and NO.

 Line 29:   marker was increased on groups - according to the content of the article, there was only one group, perhaps the authors wanted to write rats. And in general, throughout the text it is better to write "in rats from such and such a group", and not just in a group. Throughout the text, authors should more precisely and clearly articulate what they want to express.

Authors reply:

Your respected comment was completely followed in all revise paper.

Lines 34-36: The meaning of the final sentence in the abstract is not clear. The authors need the help of a professional translator.

Authors reply:

 The conclusion in the abstract was totally changed [In conclusion, living at high altitude induced hepato-renal damage, biochemical and molecular alterations, all of which may serve as a critical factor that must be taken in mind for living at high levels.]

Line 49: repetition of the abbreviation given on line 48

Authors reply:

The repetition was removed.

Lines 61-62, 81-86: English problems

Authors reply:

The introduction was totally formatted.

The introduction does not clearly state the aims of the study.

Authors reply:

 At the end of introduction the clear aims of current study were described.

Line 88: it is necessary to indicate how old the rats were and in what conditions (above sea level) they lived from birth to the beginning of the experiment.

Author reply:

The experimental condition was adjusted.

 [Rats were birthed at normal sea levels at Jeddah and housed for 2 months with average weight range from 150-170 grams, Next, rats were divided into two groups (Normal sea-level group; Group A) kept at Jeddah for extra 2 months, and  group B (High altitude group), taken to Taif region (high altitude) and maintained for extra 2 months. At the end of experimental study (2 months), rats were decapitated euthanized after anesthetization].

Line 93: English problems

Authors reply:

Corrected and the blood was taken from the orbit (retro-orbital venous plexuses).

 Line 101: reduced glutathione (GSH),- why is the word reduced  needed here?

Authors reply:  Word reduced was removed

Line 104: GPT, GOT - please explain the abbreviations

Authors reply:  Complete description for the abbreviated words was added.

Line 105: using Spectrum Co. kits - it is necessary to give an exact description of the kits

Authors reply:

The kits were from Spectrum Diagnostics, Obour City, Egypt. The details of kits were added to the revised file.

[http://www.spectrum-diagnostics.com/new/p_01_02_Enzymes.php].

 Line 114: what 260/280 ratios were obtained and considered acceptable for PCR?

Authors reply:

To examine RNA purity we measure the 260/280 ratios using Biorad spectrophotometer. If the ratio between 1 and 2 that mean RNA purity is good and can be used for cDNA synthesis.

We added this sentence: RNA samples with 260/280 ratio between 1 and 2 indicates high purity and used for cDNA synthesis

 

 Lines 118-119 - text repetition

Authors reply:

Repeated sentence was removed

 

Line 120, 121, English problems

Authors reply:

Corrected (Comparative cycle threshold (CT) values were assessed to examine the intensity and mRNA expression of the examined genes)

123, English problems

Authors reply:

 Primers names, accession number and sequence used for quantitative real time PCR in rats.

 

134, English problems

Authors reply:

A multiparametric quantitative lesion assessment was performed after 40 imaging for group A and B separately.

 

156, English problems

Authors reply:

Values with at p<0.05 are considered statistically significant for examined groups.

 

 165: English problems

Authors reply:

Corrected …….control changed by normal sea level

Line 120: According to the obtained cytological data, it can be assumed that the cell cytoskeleton changes during hypoxia. This may mean that beta-actin can change the level of expression and, if so, the authors cannot use its expression as a reference gene when performing real-time PCR.

Authors reply:

Beta actin used as internal standard and cannot be changed between groups and it is hypothesized by previous published papers. Same as serum changes.

As you know, the ^2−ΔΔCT method is more accurate for real time PCR analysis as it measure the expression of each examined gene relative to the internal beta actin standard of same sample. After that ^2−ΔΔCT method normalized the expression of examined genes as 1 for normal sea level group then divided by the relative expression of genes for high altitude group using same program and expressed as a relative fold of normal sea level group.

Lines 129-130: shift text relative to the table

Authors reply:

Shifted and can be shifted and fitted, in the final revised processing steps it will be more considered.

Table 1: you need to enter the names of the genes,

Authors reply:

The full names of genes were added down the table.

Line 138: hemoxugenase-1 - gross misprint

Authors reply:

Corrected to heme oxygenase-1

Line 140-143: HO-1- The abbreviation was not entered, English problems, sentences are written very complicated and difficult to read.

Authors reply:

This paragraph was totally formatted to be:

For immunohistochemical analysis, slices were embedded in paraffin then deparaffinised and rehydrated. They were then soaked for 15 min in 2 % H2O2 then washed in PBS to inhibit peroxidase activity. Nonspecific binding sites were blocked using 5 % bovine serum albumin.O-1 Antibody (ab13243) and Nrf2 (ab31163) Antibody were from Abcam Corporation, Cambridge, MA, USA. These antibodies were diluted to 1:500 and added to the prepared slices from liver and kidney tissues. Sample slides were incubated overnight at 4 °C. The slides were then washed three times with PBS and incubated with a 1:2000 dilution of biotin-conjugated secondary antibody (cat# sc-2040). These were developed using 3,3-diaminobezidine tetrahydrochloride and counterstained with hematoxylin [29].

 

Lines 145-146: information (objective 40)/marker/organ/animal (40 HO-1 145 images and 40 Nrf2 images/organ/group) is hard to understand.

Authors reply:

New short description was added.

[In a five non-overlapping, randomly selected microscopic fields different snapshots were taken (8 shots per field for 5 different fields) for each gene to examine the immunoreactivity between group A (sea level) and B (high altitude)].

Lines 147- 148: It is not clear what +Ve and -Ve are. Why is the ";" sign used after the word scale?

Authors reply:

The way to explain the immunoreactivity was changed.

[These images were examined to measure the degree of immunoreactivity for the changes on the expression of Nrf-2 and HO-1].

Tables 2 and 3, and 4, and 5, and Figures 1, and 2: the authors must explain what the letters a and b in the table mean

Authors reply:

All your respected comments were explained in Table 2, 3, 4, and 5, and figures 1 and 2.

Values with different letters show significant differences between the group A (normal sea level) and B (high altitude) at p<0.05.

Line 174: “per each experiment.” – you had only one experimental group,  did you mean “per each group”? There is not a single * sign indicating significant differences in the table.

Authors reply:

We corrected each to be per group and the sign * was removed and new description was added.

[Values with different letters show significant differences between the group A (normal sea level) and B (high altitude) at p<0.05].

Line 177: why only MDA, catalase, and SOD are listed, but what about NO and GSH?

Authors reply:

Sorry this unintentional mistake, we corrected it.

Line 181: same as for line 177

Authors reply:

Sorry for this mistake, we added missed words.

Line 189: when writing IFN gamma, sometimes a hyphen is used, and sometimes for some reason not. Same for other genes (EGLN-2, Nrf-2). Check the whole text, please

Authors reply:

All we unified.

Table 3: Correct the spelling of all symbols.

Authors reply:

All were corrected

 

 Line 189: the abbreviation HA was not introduced

Authors reply:

The complete name was added (high altitude; HA).

Line 190: inflmmations

Authors reply:

Corrected: inflammations

Line 191: taif region. – probably Taif region.

Authors reply:

All sentence was corrected to be: This confirm that high altitude (HA) is a stress factor that mediate inflammations and body response in subject live at Taif region.

Lines 204-211: the first sentence is very confusing. Please describe everything about Figure 1 first and then about Figure 2.

Authors reply:

The paragraph was totally changed to be:

The data presented in figure 1, showed the changes in gene expression of liver on rats lived at sea level (group A) and high altitude (group A). There were significant up-regulation on the mRNA expression of hepatic VEGF, type 1 collagen, Cox-2, TNF-α, and iNOS in Group B (Taif region; high altitude) compared to group A lived at normal sea level. However, there was a down regulation in the expression of AMPK (Figure 1). 

Regarding the effect of high altitude on the expression of renal genes, data in figure 2 showed that there were considerable up-regulation in the expression of renal EPASI, CMYC, HIF-α, and EGLN-2 in high altitude group (Group B) compared with normal sea level group (group A). There was no changes in the VHL mRNA expression between groups.

 

Line 207: Tere were

Authors reply:

Corrected

 

Line 210: “There was no changes in the VHL mRNA expression between both high altitude and normal sea level.” - Please read and think about what you have written. It turns out that you characterize the sea level by the level of gene expression. And this problem is all over the text. It is necessary to make the text understandable for the reader.

Authors reply:

We mean that the expression of VHL is not changed between groups.

 

Lines 235-236 and 258-259: oxidative tress  quantitative real - improve, please;

Authors reply:

Corrected in both figures (Figure 1 and 2) and changes to be:

Impacts of high altitude on hepatic genes associated with oxidative stress, and hypoxia in liver using quantitative real time PCR.

Bars with different letters show significant differences between the group A (normal sea level) and B (high altitude) at p<0.05

Lines 235-237, 258-260: Figures 1 and 2 show not Impacts of high altitude on hepatic genes, but Impacts of high altitude on hepatic genes expression; hepatic genes cannot be associated with biomarkers - please check the meaning of the sentence and the overall correctness of the translation. 10 different rats per treatment - the word treatment can only be used in relation to one group. The label of the Y axis is not true, it reflects the general content of the figure, but not what the numbers on the Y axis indicate.

Authors reply:

The description in both figures was changed to be:

Bars are the densitometric analysis for the expression of examined genes for 10 different rats per group. Bars with different letters show significant differences between the group A (normal sea level) and B (high altitude) at p<0.05.

Number in Y axis means the densitometry values for examined genes relative to normal sea level.

Line 265: “hepatic tissue of high attitude group” - All phrases like this should be corrected in the text of the entire manuscript. For example, this particular sentence should be corrected to “hepatic tissue of  rats from high attitude group”

Authors reply:

The sentence was changed to be:

The hepatic tissue of rats from high attitude group showed marked congestion of central vein, nuclear pyknosis, swelling, vacuolar degeneration and areas of hepatocytes necrosis (Fig. 3C).

 

Lines 263-276: It is customary to refer to the figures in order - first 3a, then 3b, then 3c and then 3d. the same for figure 4/

Authors reply:

All were corrected as you suggested in results description and figure legend.

Line 302: attitude

Authors reply:

Corrected and changed to altitude in all explained positions.

 

Line 304: stained liver of the high attitude group

Authors reply:

Corrected and changed.

 

Line 307: attitude group

Authors reply:

Corrected and changed.

Legend for Fig.4 also contains many mistakes

Authors reply:

Corrected and changed as the same for figure 3.

 

Line 338:” Lesion scoring as described by H& E“ - I guess H&E can't describe anything

Authors reply:

We mean by lesion scoring the changes reported in normal histology and pathology of hepatic and renal cells. We changed it to histiopathological changes.

 

Lines 346-351: many mistakes

Authors reply:

The paragraph was changed to be:

The current study confirmed the alterations reported when rats lived at 2 different locations for 2 months (normal sea level at Jeddah and at high altitude area in Taif). A decrease in liver and kidney biomarkers, and an increase in oxidative stress marker were reported in rats lived at high altitude at cellular and molecular levels. The expression of hepatic Nrf2, HO-1, VEGF, type 1 collagen, Cox-2, TNF-α, and iNOS together with renal EPASI, CMYC, HIF-α, and EGLN-2 were all up-regulated. There were more changes on the morphology of liver and kidney in high altitude group compared to normal sea level group. In parallel, living at Taif area induced a state of inflammation as reported by the increase in IL-1b, TNF-a and IFNg levels in high altitude compared to normal sea level.

 

 Line 352, English problems

Authors reply:

The sentence was changed to be:

Although the percentage of oxygen in inspired air remains constant at different altitudes, the partial pressure of inspired oxygen and thus the driving pressure for gas exchange in the lungs drops as atmospheric pressure falls at higher altitudes.

365: English problems

Authors reply:

The complete sentence was changed to be:

Physiologically, this impairs the liver's ability to detoxify drugs and decreases the sensitivity of liver cells to act well and this explains the reported alteration in liver activity in group lived at Taif area.

Line 418: why it is written IFN alfa?

Authors reply:

Corrected to gamma

Line 420: Why do you think there is only one pathway involved?

Authors reply:

No, of course there are several pathways are involved. We changed it.

Line 421: English problems

Authors reply:

All sentence was changed and written in small sentences.

[There are many molecular events and pathways that are associated with living at high altitude; our findings showed that there were significant upregulation of hepatic VEGF, type 1 collagen, Cox-2, TNF-α, and iNOS in the high altitude group compared with the normal sea level group, and there was a significant downregulation of the hepatic AMPK mRNA expression. In parallel, high altitude group showed significant up-regulation of renal EPASI, CMYC, HIF-α, and EGLN-2 levels compared with normal sea level group, and there was no changes in the VHL expression between examined groups.].

Line 424:  high altitude groups - why do you write that there were several such groups in the experiment?

Authors reply:

Your comment was respected and the word written in singular (altitude)

Line 500: in many rat organs - You analyzed only two organs

Authors reply:

Corrected

Line 506: “Cautions must be taken, and more health care must be directed to people living at high altitudes.” - This phrase is not very correct, since the local population, permanently living at an altitude of 2500 m above sea level, is unlikely to have such consequences as described in the experiment. Rather, this applies to tourists who come to rest.

Authors reply:

Your respected comment was followed and the sentence and word cautions was omitted.

Make gene symbols throughout the text in italic

Authors reply:

All genes were corrected in italic in all the revised paper.

Reading the manuscript is difficult because the authors use numerous abbreviations, many of which have never been deciphered. There is also no list of abbreviations.

Authors reply:

The paper has been edited by MDPI editing and proofing service. Please see attached certificate.

A list of abbreviations were added after conclusion.

New collective figure (Figure 5 was added in conclusion section).

 

The text contains a significant number of typos and translation errors. This list of comments does not list all errors and typos.  Also, there are a lot of phrases in the text that incorrectly reflect the meaning of what the authors wanted to say. It is necessary to carefully check the content of the text and edit the manuscript with the help of a professional translator or native speaker.

Authors reply:

Sorry for this mistake.

The paper has been edited by MDPI language editing service. Please see attached Certificate of editing.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have significantly improved the text of the manuscript. However, there are still a few comments on the text. In addition, some of the responses raised additional questions that require clarification.

Lines 93- 94: In one sentence, two sentences are mixed, which reads very strange.

Lines 121-122: “RNA samples with a 260/280 ratio between 1 and 2 indicates high purity and were used in cDNA synthesis” –  Since RNA samples with a ratio of 260/280 in the range of 1.8-2.0 are usually considered purely isolated, the authors' answer to the question raises the need to ask additional questions in order to understand the quality of real-time PCR analysis. Authors should describe in detail in the text of the manuscript how the RNA was isolated, whether they used DNase to remove residual DNA, whether they performed control PCR reactions to confirm that the amplification was carried out on RNA, but not on DNA. How were the primers selected – within one exon or across an intron, or across the border of two exons? In Table 1, authors should indicate the length of the amplified fragments and the annealing temperature of the primers.

Line 127: “The internal gene β-actin was employed as a control“ –  probably you wanted to write “The gene β-actin was employed as an internal control“. In addition, the authors' response that "Beta actin cannot be changed between groups" is not satisfactory. The authors must provide evidence that β-actin is transcribed in equal amounts in the liver and kidneys of control and experimental rats. To clarify the point of view of the reviewer and to explain why the reviewer revisited this issue, after the comment on Figure 5, references are given to articles that show unstable expression of the beta actin gene in various experiments. Since, in the manuscript under review, hypoxia caused structural changes in liver and kidney tissues, it is very likely that the beta actin gene could also change its transcription level in experimental animals compared to control rats.

Lines 144-145: “A multiparametric quantitative lesion assessment was separately performed on 40 images each for group A and B as shown in Table 5.” – Did you mean “40 images for each group”?  In addition, it is necessary to indicate how many animals in each group were tested.

Line 203: Why is the word “groups” given in the plural?

Line 282: “polyhedral cells hepatocytes” – did you mean to write “polyhedral cells (hepatocytes)“?

Line 358: “for 5 samples/group”.  - Please indicate from how many different animals the samples were tested

 Line 365: ”markers for rats lived that lived at” -  this text needs correction

Figure 1: Designations a and b for the AMPK gene are swapped. In addition, you need to make correction to the signature of the “Normal sea leve”

Figure 2: Since designations a and b are tied to a certain group, it is not correct to designate the same letter a for the results obtained for different groups of rats. It is probably better not to indicate the absence of differences in the level of transcription of the VHL gene. In addition, you need to make corrections to the signature of the “Normal sea leve”

 Figure 5: Why is the word “damage” written twice?

 

Links to articles showing unstable expression of the beta actin gene in various experiments:

19. Leduc, V. Legault, D. Dea, J. Poirier, Normalization of gene expression using SYBR green qPCR: A case for paraoxonase 1 and 2 in Alzheimer's disease brains, J. Neurosci. Methods 200(1) (2011) 14-19. - (…Actb expression in human brain affected by Alzheimer’s disease was not stable)

Fedoseeva  L. A., Shevelev O. B., Kolosova N. G., Dymshits  G. M. MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain. Biochemistry (Moscow), 2014, Vol. 79, No. 7, pp. 706-716. – “Using phage based normalization, we analyzed mRNA levels of three popular housekeeping genes coding β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein L30 and showed high variability in their expression patterns during rat brain development, indicating that they should not be used as controls in gene expression studies of the developing brain either individually or in combination.”

221. Chang, C. Ling, M. Yamashita, N. V. Welham. Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury. Anal. Biochem. 406 (2010) 214–221. – “A number of genes, such as Gapdh, B2m, and Actb, are commonly utilized as reference genes for qRT-PCR data normalization across a wide variety of experimental systems; however, many have been demonstrated to be unstable under certain conditions [10–15]. Moreover, several studies have shown that a reference gene that is stable in one experimental system might be unstable in another [11,16,17].” “Actb, B2m, and Gapdh, three candidates commonly employed (but previously unvalidated) as reference genes in vocal fold and other mucosal injury models [32–39,51,52], were also unstable postinjury.” Z. Chang, C. Ling, M. Yamashita, N. V. Welham. Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury. Anal. Biochem. 406 (2010) 214–221. - “Gapdh and Actb were ranked as the two least stable genes by stepwise ranking of expression stability using the geNorm algorithm”.

W.A. Schulz, P. Eickelmann, C. Hallbrucker, H. Sies, D. Haussinger, Increase of beta-actin mRNA upon hypotonic perfusion of perfused rat liver, FEBS Lett. 292 (1991) 264–266

114. Dheda, J.F. Huggett, S.A. Bustin, M.A. Johnson, G. Rook, A. Zumla, Validation of housekeeping genes for normalizing RNA expression in real-time PCR, Biotechniques 37 (2004) 112–114. – “It is now clear, however, that in most experimental situations the use of conventional reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and _-actin, is inappropriate due to their variability [1,2].” ([1] J. Huggett, K. Dheda, S. Bustin, A. Zumla, Real-time RT–PCR normalisation: strategies and considerations, Genes Immunol. 6 (2005) 279–284. [2] S.A. Bustin, T. Nolan, Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction, J. Biomol. Tech. 15 (2004) 155–166.)

Author Response

Dear respected reviewer, great thanks for your comments and we have revised the manuscript based on your respected points. The corrections are in red color in submitted files

The authors have significantly improved the text of the manuscript. However, there are still a few comments on the text. In addition, some of the responses raised additional questions that require clarification.

Lines 93- 94: In one sentence, two sentences are mixed, which reads very strange.

Authors Reply: The sentence was corrected.

Lines 121-122: “RNA samples with a 260/280 ratio between 1 and 2 indicates high purity and were used in cDNA synthesis” –  Since RNA samples with a ratio of 260/280 in the range of 1.8-2.0 are usually considered purely isolated, the authors' answer to the question raises the need to ask additional questions in order to understand the quality of real-time PCR analysis. Authors should describe in detail in the text of the manuscript how the RNA was isolated, whether they used DNase to remove residual DNA, whether they performed control PCR reactions to confirm that the amplification was carried out on RNA, but not on DNA. How were the primers selected – within one exon or across an intron, or across the border of two exons? In Table 1, authors should indicate the length of the amplified fragments and the annealing temperature of the primers.

Authors reply:

New paragraph was added in methodology about RNA extraction and qRT-PCR

[Total RNA was extracted from liver and kidney tissues of group A and B using RNeasy Mini Kit (Cat# 74104,  Hilden, Germany). RNA purity was measured using the A260/A280 ratio. RNA samples with A260/A280 ratio between 1.8 and 2 indicated high purity and were used in cDNA synthesis. The complementary DNA (cDNA) was synthesized with the HiSenScript kit by mixing 10 µl of 2 RT reaction solution, 1 µl of the enzyme, and 1 µg of total RNA and made up to final volume of 20 µl with RNase free water. The reverse transcription reaction was made by incubating the mixture at 50°C for 30 minutes and then at 85°C for 10 minutes to inactivate the enzyme. The qRT-PCR was carried out on the PCR thermal cycler machine (iQ5 Real-time PCR, Bio-Rad, USA) using Quanti Fast SYBR Green PCR kit. The information of primers, sequence, and product size are listed in Table1. Each PCR reaction consisted of 1 μl cDNA, 10μl SYBR Green PCR Master Mix (Quanti Tect SYBR Green PCR Kit, Qiagen, Valencia, CA, USA), along with 1 μM of forward and reverse primer for each examined gene and nuclease free H2O to a final volume of 20μl. Reactions were run and analyzed in iQ5 Real-time PCR, Bio-Rad machine. Real-time PCR conditions were: first denatured at 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 seconds (second denaturation), then annealing as shown in table 1 for 60 stage. The critical threshold (Ct) of the target gene was normalized with quantities (Ct) of the housekeeping gene (β-actin), using the formula x = ^{2 − ΔΔ}Ct, where there is x = fold difference.].

 

DNase digestion is not required with RNeasy Kits since RNeasy silica-membrane technology efficiently removes the DNA without DNase treatment as indicated in the kit manual.

Primers designed base on the gene accession number and selected within same exon.

 

 

Line 127: “The internal gene β-actin was employed as a control“ –  probably you wanted to write “The gene β-actin was employed as an internal control“. In addition, the authors' response that "Beta actin cannot be changed between groups" is not satisfactory. The authors must provide evidence that β-actin is transcribed in equal amounts in the liver and kidneys of control and experimental rats. To clarify the point of view of the reviewer and to explain why the reviewer revisited this issue, after the comment on Figure 5, references are given to articles that show unstable expression of the beta actin gene in various experiments. Since, in the manuscript under review, hypoxia caused structural changes in liver and kidney tissues, it is very likely that the beta actin gene could also change its transcription level in experimental animals compared to control rats.

Response:

1. We mean beta-actin is the internal standard for each sample. Not as a control for the experiment, because the control is normal sea level and changes in gene expressions.
2. Expression of each examined gene was normalized to its internal standard beta actin gene expression based on formula x = ^{2 − ΔΔ}Ct that automatically show the fold change of each gene relative to control group (sea level).

 

Lines 144-145: “A multiparametric quantitative lesion assessment was separately performed on 40 images each for group A and B as shown in Table 5.” – Did you mean “40 images for each group”?  In addition, it is necessary to indicate how many animals in each group were tested.

Authors reply:

A multiparametric quantitative lesion assessment was separately performed on 40 images (8 images/5 rats per each group) as shown in Table 5.

Line 203: Why is the word “groups” given in the plural?

Authors reply:

Thanks for comment we have corrected and removed s

 

Line 282: “polyhedral cells hepatocytes” – did you mean to write “polyhedral cells (hepatocytes)“?

Authors reply:

We corrected it to be:

Histology of the hepatic tissue of the control group (Figure 3 A) shows the presence of intact polyhedral hepatocytes arranged in a cord-like pattern radiating from central vein, and each cord is separated from the others by hepatic sinusoids

 

Line 358: “for 5 samples/group”.  - Please indicate from how many different animals the samples were tested.

Authors reply:

We corrected it to be:

Values are mean ± SE for 5 rats/group

 

 Line 365: ”markers for rats lived that lived at” -  this text needs correction

Authors reply:

Two words (that lived) were removed, the sentence became:

These alterations at biochemical, cellular, and molecular levels through the decrease in liver and kidney biomarkers and an increase in the levels of oxidative stress markers for rats lived at high altitude compared to normal sea level control group

Figure 1: Designations a and b for the AMPK gene are swapped. In addition, you need to make correction to the signature of the “Normal sea leve”

Authors reply:

All are corrected. The signature was corrected.

Figure 2: Since designations a and b are tied to a certain group, it is not correct to designate the same letter a for the results obtained for different groups of rats. It is probably better not to indicate the absence of differences in the level of transcription of the VHL gene. In addition, you need to make corrections to the signature of the “Normal sea leve”

Authors reply:

Designations for VHL gene were removed. The signature was corrected.

 Figure 5: Why is the word “damage” written twice?

Authors reply:

The extra word was removed.

 

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

The authors have made corrections, but the answers to questions regarding real-time PCR are not completely satisfactory.

• The RNeasy Mini Handbook says that “Generally, DNase digestion is not required with RNeasy Kits since RNeasy silica-membrane technology efficiently removes most of the DNA without DNase treatment. However, further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundance target).” The qRT-PCR used in this study is able to amplify a low-abundance target. Accordingly, the authors had to test the quality of the isolated RNA for the presence of DNA residues and show that the amplification proceeds on the RNA without a significant DNA contribution.Either the authors had to use primers that make it difficult to amplify the DNA fragments, i.e.primers must be chosen across the long intron, or across the border of two exons.
• In response to the question about the stability of beta actin expression, the authors correctly describe the principle of the method, but do not take into account that the method can be used only if the expression of the reference gene is equal in rats of the experimental and control groups.The authors need to either show that the expression of beta actin in the kidneys and liver of rats does not change when rats are transferred to high altitude conditions, or use the geometric mean of several reference genes.

Lines 359-360: There is no verb in this sentence.

Author Response

Reply to reviewer comments (cimb-1646502)

Dear Respected reviewer:

Following your wishes, we have now changed this manuscript to be more suitable for publication. With all due respect to the reviewer, your revisions and suggestions enabled us to improve the paper quality. The following are our point-by-point responses to each of your comments:

Kindly note that all revisions and modifications are in red in the manuscript.

The RNeasy Mini Handbook says that “Generally, DNase digestion is not required with RNeasy Kits since RNeasy silica-membrane technology efficiently removes most of the DNA without DNase treatment. However, further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundance target).” The qRT-PCR used in this study is able to amplify a low-abundance target. Accordingly, the authors had to test the quality of the isolated RNA for the presence of DNA residues and show that the amplification proceeds on the RNA without a significant DNA contribution. Either the authors had to use primers that make it difficult to amplify the DNA fragments, i.e. primers must be chosen across the long intron, or across the border of two exons.

Authors Response:

Thanks for your revisions and suggestions.

We rerun the PCRs using the followings:

1. We used (non-template controls), and RT-PCR + and RT-PCR -, respectively)
2. (RT-PCR -) Negative control which consists of all the reagents of the cDNA synthesis (including the RNA sample) without reverse transcriptase (enzyme) and all samples did not show any product of amplification
3. (RT-PCR +) positive control to see cDNA amplification from the RNA.

4. This proved that there was no primer-dimer or DNA contamination in our RNA sample

1. The quality and quantity of the extracted RNA were determined by Nanodrop (Quawell, USA). Samples of 1.8 or more A260 / A280 RNA were used. In addition, the quality of the extracted RNA was confirmed with 2 % agarose electrophoresis.

 

In our laboratory, we make the following strict conditions to avoid contamination

1. Ensure no contamination is designated and use distinct areas for sample preparation, PCR setup, and post-PCR analysis. To avoid contamination from old amplicons, we set up the stations on different benchtops, one for pre-PCR (PCR reaction setup only) and the other for post-PCR (purifying PCR-amplified DNA, measuring DNA concentration, running agarose gels, and analyzing PCR products).
2. Restrict equipment to these areas. Keep the PCR machine and electrophoresis apparatus in the post-PCR area.
3. Prepare and store reagents for PCR separately and use them solely for their designated purpose. Aliquot reagents in small portions and keep them in either location based on their use in pre-PCR or post-PCR applications. Store the aliquots separately from other DNA samples.
4. Use separate sets of pipettes and tips, lab coats, glove boxes, and wastebaskets for the pre-PCR and post-PCR areas.
5. Use pipettes and pipette tips with aerosol filters dedicated for DNA sample and reaction mixture preparation.

 

 

In response to the question about the stability of beta actin expression, the authors correctly describe the principle of the method, but do not take into account that the method can be used only if the expression of the reference gene is equal in rats of the experimental and control groups. The authors need to either show that the expression of beta actin in the kidneys and liver of rats does not change when rats are transferred to high altitude conditions, or use the geometric mean of several reference genes.

 

Authors Response:

Thank you for your comment.

1.  According to your advice, we repeated and re-checked the stability of standard genes using multiple internal control genes in this experiment; we try to assess the most stable reference genes using multiple algorithms. then select the genes that are most stable with all these algorithms
2. We use both GAPDH and β -actin among different samples and control one. We found that the output of β -actin and GAPDH among different samples are more stable, so we continue using β -actin and GAPDH see table 1.
3. In addition, we used geNorm method (Hellemans et al. 2007) to calculate the target stability between the different conditions). The geometric mean of the two reference genes was used.

We calculated the fold-differences using the ddCt method that is usually expressed as a range, which results from incorporating the standard error of the ddCt value into the fold-difference calculation. Please see this attached link for a detailed description.

Please copy and paste this down link in chrome for more details.

 (chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/viewer.html?pdfurl=https%3A%2F%2Fwww.science.smith.edu%2Fcmbs%2Fwp-content%2Fuploads%2Fsites%2F36%2F2015%2F09%2FAnalyzing-your-QRT-for-relative-2%255E-%25E2%2588%2586%25E2%2588%2586Ct.pdf&clen=806571&chunk=true)

 

Lines 359-360: There is no verb in this sentence.

Authors Response:

We modified it according to your comment (please, see the highlighted red-colored lines 360-365).

[The current study confirmed feedback of living at high-altitude level in Taif compared to normal sea level at Jeddah for 2 months. We confirmed the changes in liver and kidney biomarkers at biochemical, cellular, and molecular levels. There were an increase in the levels of oxidative stress markers for rats living at high altitudes compared to the normal sea level control group, and a decrease in Nrf2 and HO-1 immunohistochemistry in rats lived at high altitude.].

Author Response File: Author Response.docx