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This is a review for the article “Bioinformatics-based identification of RAS disequilibrium involved in post-hemorrhagic shock mesenteric lymph return mediated acute kidney injury in mice.”. authors used bioinformatics to identify pathways shared between PHSML return and acute kidney injury and claimed to find that RAS system in involved in both injuries along with some other pathways. Authors then conducted a mouse animal model of PHSML return and studied the effect of mesenteric lymph duct ligation (MLDL).
Authors found that the RAS system was activated in kidney tissues, and an imbalance of AngII/Ang (1-7) resulted from this injury. The authors used inhibitors and the Ace2 -/- model to confirm this pathophysiological relationship.
In general, the manuscript was written in fair English. Some rephrased sentences are mentioned below.
The introduction was fine. Enough background and related to the topic.
Methods were described well and had no comments on them.
Comments on results and figures are together below:
Results for the bioinformatics section are straightforward and understandable.
Line 145-147: Renal tubules narrowed in shock only kidneys, as claimed from figure 2. But in later figures with immunostaining for the shock, only animals in figures 3 and later one can notice tubule dilatation in the shock kidney slides. Can authors clarify this point?
Line: 147-148 (Renal tubules were narrowed in the kidneys, and acute hemorrhage and resuscitation were performed). This sentence needs rephrasing.
Figure 2:
The histology fields shown do not show clear histological differences between different conditions. Furthermore, authors mentioned in the results section that their injury model resulted in (the mesangial cells were proliferating, the basement membrane was ruptured, glomeruli were compressed, renal tubular epithelial cells showed high edema, and even extended into the vessel cavity). Kindly use marking objects, e.g, asterisks, arrowheads, arrow, etc, to show the mentioned pathological features.
A magnification scale would be good to be available in the histology pictures for this figure and the remaining figures, too.
Figure 3:
The authors used 4 different antibodies in this figure, that was mentioned in the legend but not next to each panel of the picture. I have to go back and forth between the legend and the figure to be able to know which staining is for which picture. Kindly correct this on the figure by writing each immunostain next to the respective panel. Scale bars should also be added.
The figure contains bar charts labelled with (F), but nothing is mentioned about them in the legend, and with which the concentrations and ratios were calculated.
Figure 4:
Same comments as figure 3 about the labeling of picture panels with respective immuno-stain. Also, the scale bars.
Figure 6:
This comment is about this figure and the whole part of this experiment in the results section. The authors used the ace-/- mouse strain and claimed in the results section the following:
(174 Inhibition of the ACE2/Ang (1-7)/MasR axis partially abolished the MLDL beneficial effects on
175 mice after hemorrhagic shock
176 The Ace2 knockout and Ang II and A-779 administrations were executed as shown in Figure 6A,
177 and induced more severe pathological injury in the kidneys of mice following hemorrhagic shock
178 plus MLDL)
If the inhibition of the ACE2/Ang (1-7)/MasR axis partially abolishes MLDL beneficial effects, then the authors need to show a comparison of this with the following conditions:
• Shock only
• Shock+MLDL
• Shock+MLDL+inhibition of ACE2/Ang (1-7)/MasR models (i.e., and treated with Ang II or A-779)
And this was not shown in Figure 6.
Again, the reader cannot notice the induction of more severe injury between the H&E slides. Can authors elaborate more and show the signs of the more severe injury on the slides? Kindly use marking objects like arrows … to justify their results.
Furthermore, authors stained slides for ACE2, and the slide from the ace2-/- knockout strain shows slight positivity for the ACE2 immunostaining. Can authors justify these results? I mean, why do ace2 -/- mice have any positivity for ACE2 immunostaining?
Figure 7:
The legend is completely wrong and was just copied from Figure 6. The pictures and the charts are about the expression of ACE, AT1R, and Ang levels, and the legend is for ACE2, MasR, and Ang (1-7).
In discussion
287 In addition, ACE2-/- mice showed more severe histopathological
248 changes than mesenteric lymphatic ligation mice, even with lower expression of MasR and Ang
249 (1-7). These results suggest that MLDL alleviates the imbalance of ACE/ACE2 and pathological
250 changes in the kidney; however, the mechanism by which MLDL regulates the components of
251 RAS is not clear.
But figure 6 and the bar chart in it do not show that Ace2-/- animals with shock and MLDL had lower MasR expression.
One major concern or limitation of this study is that AT2R (Angiotensin II Type 2 Receptor) was not studied. This either should be done or mentioned as a limitation in the discussion.
Another comment is that the authors used only the immunostaining technique in their expression investigations. ELISA, western blotting, and/or real-time PCR techniques were not used.
Overall, the English in the article is adequate. However, word choice can be improved in a few areas. For example, "less severe" instead of "lighter" in line 149. Again, "lightly" is used incorrectly in lines 163 and 164.
The introduction and discussion are supported by adequate references. Overall, the article flows in a logical fashion from one section to the next.
In this article, the authors test the hypothesis that the balance between ACE and ACE2 expression explains the protective effect on kidneys of mesenteric lymphatic duct ligation following hemorrhagic shock. They tested this hypothesis through the use of a complete ACE2 knockout mouse model as well as different drugs that affect the ACE and ACE2 signaling axes.
The research question is well-defined, and enough background information is provided to demonstrate the current gap in our knowledge and the relevance of investigating ACE/ACE2 activity following mesenteric lymphatic duct ligation in hemorrhagic shock.
The authors should improve their description of the experimental design by stating the number of mice used in each group for every experiment in the results section.
Major concerns with this study lie in the presentation and robustness of the data, the validity of the findings, and the conclusion that the authors draw.
The figures and figure legends as they are currently displayed and written are difficult to follow, and they should be made clearer.
Here are my major comments:
1. Line 128: Please specify the name of the gene expression database used here in the Results section.
2. Lines 140-141: Specify that these are human samples from COVID-19 AKI patients. The identity of the control group should also be clarified. Are these COVID-19 patients without AKI? Are they general patients without AKI?
3. Figure 2 is likely not necessary to be included, as it does not represent new data. It is widely known that hemorrhagic shock causes AKI, and the protective effect of mesenteric lymphatic duct ligation has already been published. These figures can be moved to a supplementary data folder, or they may not need to be included in the manuscript.
4. Lines 154-155: The authors should specify whether they are comparing gene or protein expression in the text. (It is protein expression based on immunohistochemistry staining of kidneys.) They also state that this is a histologic comparison.
6. Figure 3: Please label each row of micrographs with the marker being stained.
7. Many micrographs throughout the figures are poor-quality images and appear blurry, making it difficult to ascertain the findings that the authors describe. In addition, higher magnification images of the glomeruli and tubules would help in demonstrating the findings.
8. Line 176: What is A779, and why is it relevant to the experiment?
9. The authors use histopathologic data as the basis for their assessment of kidney injury in this study. Given that the mice were euthanized 4 hours after resuscitation, it is understandable that serologic markers of kidney injury (blood urea nitrogen, creatinine) may not be elevated at this early time point. (The authors should state this in the discussion.) However, the micrographs provided by the authors in the figures are inadequate to demonstrate their findings of kidney injury. If purely histologic data are used, then the authors should include some semiquantitative assessment of the histologic injury. For example, the percentage of the microscopic fields affected by tubular injury. In addition, staining for a tubular injury marker such as KIM-1 is important to add robustness to the data.
10. Assessment of 3 microscopic fields per sample is inadequate. More fields should be assessed (at least 8-10 per sample).
11. The number of mice used must be clearly stated throughout the manuscript. Use of just 3-4 mice per group throughout the study raises questions about the robustness of the findings.
12. The figure legends must be more descriptive, especially for the immunohistochemistry staining. As currently written, they are difficult to follow.
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