Effect of Everolimus versus Bone Marrow-Derived Stem Cells on Glomerular Injury in a Rat Model of Glomerulonephritis: A Preventive, Predictive and Personalized Implication

Round 1

Reviewer 1 Report

I would like to congratulate the authors for the manuscript entitled “Everolimus versus bone marrow-derived stem cells on glomerular injury in a rat model of glomerulonephritis: a preventive, predictive and personalized implication”. They made a great effort to assess the impact of two therapeutic approaches for treating an acute rodent model of glomerulonephritis. However, I do have some major comments that I would like to address.

 

Major comments:

 

Comment 1, I have major concerns about the design of the present study. Why did authors choose to test Everolimus for the treatment of an acute model of glomerulonephritis and compare it to bone marrow-derived stem cells (BMDSCs)? The main limitation for Everolimus translation to clinical practice are its adverse effects on podocytes and the consequent development of proteinuria. It can induce de novo proteinuria or aggravate previous proteinuria. Therefore, Everolimus is not used in most glomerular diseases (IgA nephropathy, membranous nephropathy or other types of proliferative glomerulonephritis). Moreover, it would be difficult to tell in a patient receiving Everolimus if proteinuria increases due to the drug or the progression of the primary glomerular disease.

 

Comment 2, In line with the previous comment, I miss a steroid-treated rat group. Steroids are the main treatment for acute glomerulonephritis and would have provided an adequate control in this experiment.

 

Comment 3, I think authors should try to clearly define the objectives of the present study in the manuscript and explain the impact of these findings in future therapeutic approaches. What did the authors expect to observe, and did the findings correlate with these expectations? Probably an important result is the minimum protective effect exerted by BMDSCs compared to Everolimus. As the authors describe in the Discussion, it may be related to the dosing and the administration route and give us insights for future experiments.

 

Comment 4, In the Results section, showing bar graphs with mean and SEM is not adequate and does not help understand the data distribution. Authors should at least show SD instead of SEM. Moreover, if they do not want to use dot plots, Tukey’s box plots are an adequate option.

 

Comment 5, Why did authors check TBARs and MCP-1 in urine, considering they had kidney samples for IHC and protein expression analysis? Urine does not always reflect what is happening in kidney tissue, although findings are afterwards reinforced by flow cytometry. Did the authors check for inflammation or oxidative stress in kidney sections?

 

Comment 6, I recommend the authors to organize Figure 1 in panels A and B. What are the authors showing in the top panel? When evaluating apoptosis and necrosis in the kidney using flow cytometry, did the authors identify the different types of kidney cells or show overall apoptosis (total renal cells)?

 

Minor comments:

 

Comment 7, Animal research is still a sensitive topic. Therefore I would recommend using the word euthanized instead of terminated in the Methods section “…rats were placed in 102 metabolic cage for 24 hour urine collection before being terminated to collect blood and 103 kidney…”.

 

Comment 8, In the Methods section, please explain which items were evaluated in H&E sections and how. Did the authors use a semi-quantitative scale?

 

Comment 10, In the Methods section “…Cells were then treated with reagents…” is not an accurate sentence. Consider rewriting it “…Cells were treated with fluorochrome-conjugated antibodies of interest according to manufacturers’ instructions…”.

 

Comment 11, “…mouse bone marrow cells was reported to differentiate into glomerular cells that using transgene enhanced green fluorescence protein (EGFP) mice as bone marrow donors…” The sentence is impossible to understand. Consider rewriting it.

 

Comment 12, English language should be thoroughly reviewed in the manuscript. Some sentences are hard to read or impossible to understand, especially in the Introduction section. Please revise carefully.

Author Response

First, we would like to thank the reviewer for his/her helpful and instructive comments. We have revised our manuscript according to his/her suggestions. All our changes are highlighted in yellow in the revised version of the manuscript. Below is our response to the questions raised by the reviewer as follow:

Comment 1: the reviewer raised an excellent point.  We understand that long term use of everolimus might have deleterious rather than protective effect clinically. Thus, our intention was to test the protective effect of BMDSCs on experimental model of glomerulonephritis and  we used everolimus as  an immune-regulator that have previously shown to reduce renal injury in hypertensive and renal injury rat models. We have revised manuscript to clearly point out the reviewer concern. 

Comment 2: We totally agreed with the reviewer; however, we knew that steroid would improve glomerulonephritis and our main focus was to test the protective effect of BMDSCs. when injecting BMDSCs failed to provide renal protective effect, we decided to determine effect of everolimus based on the clinical data that showed it might have deleterious renal effects. Surprisely, everolimus provided a reno-protective effect in acute model of glomerulonephritis which might be due to its immuno-regulatory effect and this did not exclude the potential adverse effect of using it chronically as it is known to increase proteinuria and could exacerbate renal injury. The reviewer concern has been clearly pointed out in the revised manuscript. 

Comment 3: As the reviewer suggested, we revised the introduction to clearly indicate that our aim is to investigate renal protective effect of BMDSCs giving the fact the eveolimus might have deleterious effect on the kidney clinically in glomerulonephritis. However, our data showed that BMDSCs did not provide renal protective effect which could be attributed to dose and frequency of administration. We have carefully revised the manuscript to clarify this point. 

Comment 4: We have revised the manuscript to include SD instead of SEM in addition to Tukey’s box plots based on the reviewer suggestion.

Comment 5: Since we always collect urine from our rat experiments to look to albumin, creatinine and protein excretion as early markers of renal injury, we also used nephrin and podocalyxin excretion as markers of glomerular damage. One kidney was used for flow cytometry analysis of apoptosis  and immune cells (T cells and macrophages )and other was used for histology and isolation of glomeruli. for more than two decades, we have used urinary MCP-1 and TBARs excretion as indicative of renal inflammation and oxidative stress, respectively in experimental models of hypertension and diabetes. However, we agreed with the reviewer that this might not necessarily reflect renal inflammation and oxidative stress at least in the current model . Assessing markers of  inflammation and oxidative stress will be addressed in renal tissue in a follow up study using higher dose of BMDSCs and or repeated  administration of the current dose in the same rat model of glomerulonephritis.  

Comment 6: We would like to thank the reviewer for his excellent suggestion. in fact, figure 1 was included to indicate that AntiThy injection specifically targeted glomeruli  and induced glomerular apoptosis. This has been clearly pointed out  in the revised version of the manuscript.  

Comment 7, We have revised the manuscript based o the reviewer suggestion

Comment 8, The reviewer raised an excellent point. We have used the expertise of our clinical pathologist Dr. Rafik Abdelsayed to determine if Antithy1 injection induced glomerular damage using H & E staining in addition to our  data presented in figure 1 showing increased glomerular apoptosis. Dr. Rafik assessed  glomerular injury and any overall renal injury in a blind fashion without a semi-quantitative analysis. 

Comment 10, Correction has been made

Comment 11, Correction has been made

Comment 12, English language has been thoroughly reviewed in the manuscript based on the reviewer suggestion. 

We hope that all points have been addressed adequately and our revision is well received.

Reviewer 2 Report

In the manuscript titled “Everolimus versus bone marrow derived stem cells on glomerular injury in a rat model of glomerulonephritis: a preventive, predictive and personalized implication” the authors Zedan et al compare the protective effects of Everolimus versus BMDSCs on Thy1 induced glomerular nephritis.

 

The biggest drawback of the study is the authors do not demonstrate the localization of the stem cells in the vicinity of the target tissue, the glomerulus. They do cite some other papers that have shown presence of said cells in the glomerular tuft and interstitium in miniscule quantities. Given the paucity of these cells in these areas, they further speculate the suppression of imflammatory cytokines and other immune modulators as the possible mechanism behind their protective role. However, given the protective effect is barely there, this study essentially produces no results of significance and therefore is not recommended for publication.

Is there any technical difficulty anticipated in using GFP BMDSCs? Wouldn’t that be a method of detecting where the stem cells migrated to after being injected into the animals?

Some other points to address to improve the quality of the manuscript:

1. P2 line 72: this sentence is meaningless.
2. Line 102: what is the rationale behind the 9-day gap?
3. Assessment of glomerular albumin permeability: the authors cite PMID 22205229 which barely contains any more information than the manuscript under review; a more appropriate citation would be PMID 21191784. Also, the language of the “Assessment of glomerular albumin permeability” section of materials and methods in the manuscript under review is almost identical to that in PMID 22205229.
4. Have the authors looked into more improved methods of measuring glomerular albumin permeability, such as found here? https://www.bristol.ac.uk/translational-health-sciences/research/academic-renal/research/glom-phys/
5. For samples preparation for analytical flow cytometry, were the kidney samples subjected to enzymatic digestion?
6. For Fig1, caspase3 western blot will strengthen the data.
7. Fig 3B, does creatinine clearance from proximal tubule lead to no change in creatinine clearance between the different groups?

Author Response

Comment 1:  Correction has been made based on the reviewer suggestion.

Comment 2: The rationale behind the 9-day gap  from Anti-Thy1 injection is based on preliminary data showed that anti-thy1 induced a significant increase in glomerular apoptosis after at least one week of the injection in rats

Comment 3:  Correction has been made based on the reviewer suggestion.

Comment 4:  Although we totally understand that there more advanced technique to assess glomerular permeability, we used well-established and previously published technique in Dr. Elmarakby lab in addition to assess nephrin expression as indirect measure of loss of glomerular barrier function. 

Comment 5:  We did not use enzymatic digestion during preparation of kidney samples for flow cytometry to prevent non-specific binding and cross-reactivity of antibody. This technique has been well established and published by Dr. Baban groups many times before. 

Comment 6:  We totally agreed with the reviewer than assessment of glomerular caspase 3 using western blotting would strengthen figure 1 data/ However all our isolated glomeruli were used for flow cytometry and assessment of glomerular permeability. Additionally, we actually  assess caspase 3 as marker of apoptosis rather than Annexin V in figure 1. We apologized for the error and correction has been made in figure 1 to reflect this change. 

Comment 7:  We did not assess creatinine clearance from proximal tubule and we only used urinary and plasma creatinine level to calculate creatinine clearance. consistent with our data, we did not expect significant change in creatinine clearance based on the short term (acute) nature of the study. 

We hope that all points have been addressed adequately and our revision is well received.

Round 2

Reviewer 1 Report

I would like to thank the authors for addressing all my comments and their honest answers. Indeed everolimus worked due to its immunosuppressant and anti-inflammatory effects, and it could be another potential drug to use in glomerulonephritis if it were not for its proteinuric effects. I only have three minor comments to add:

 

Comment 1, In the Discussion section, please reinforce the idea that everolimus worked in the early anti-Thy1-induced glomerulonephritis. It will not make a translational difference, but it is evident that the authors identified an anti-inflammatory and protective effect of everolimus in this model. It will also help clarify previous conflicting results of studies using everolimus.

 

Comment 2, Define the acronym PPPM again at the end of the Introduction section

 

Comment 3, In the first paragraph of the Discussion section, consider rewriting the following sentence “…The safety and efficacy of the mTOR inhibitor everolimus in halting the progression of glomerulonephritis requires further investigation, while BMDSCs could have a promising role in the repair of injured tissues with minimal adverse effects…”. Avoid “On the other hand” in this sentence.

 

Finally, I wish the authors the best of luck in future studies using higher doses or repeated administration of BMDSCs in the same rat model.

Reviewer 2 Report

Thank you for making the revisions to the manuscript.