Early Administration of N-Acetylcysteine Provides Renal and Cardiac Mitochondrial and Redox Protection, Preventing the Development of Cardio-Renal Syndrome Type IV Induced by 5/6NX

Round 1

Reviewer 1 Report

It is well established that individuals with CKD have a very high cardiovascular risk and are closely associated with cardiovascular diseases.

This paper focuses specifically on the pathophysiology leading to CHF in CKD (CRS-4), using a 5/6 nephrectomy (NX) animal model. It analyzes the pathophysiology of this model in the early stages of CKD development, particularly from the perspective of mitochondrial function and associated oxidative stress.

Furthermore, it demonstrates that early administration of NAC in this CKD model can prevent the onset of CRS-4, providing valuable information for the development of CKD prevention strategies in clinical medicine.

Of particular interest was the discussion on the mechanism of action of NAC in this model.

The authors explain that NAC's effects extend beyond direct scavenging of ROS; it also serves as a substrate for intracellular glutathione synthesis and enhances ROS scavenging activity through intermolecular interactions with other antioxidants.

It is highly plausible that NAC is involved not only in glutathione synthesis but also in the synthesis of per/polysulfides from intracellular cysteine, and that it relates to calcium influx into mitochondria. Therefore, future research findings from these authors warrant attention.

In any case, this study clearly demonstrates the usefulness of NAC in preventing CRS-4 and is considered a paper of great importance for clinical medicine.

As mentioned above

Author Response

R= We greatly appreciate the reviewer's comments and evaluation. We are very pleased to know that the research that we conducted is valued by the reviewer.

Reviewer 2 Report

The aim of this manuscript is to investigate the cardiac and renal protective effects of NAC in a rat model of CKD (5/6 PNX). The authors report that NAC pre-treatment in the 5/6 PNX model attenuates cardiorenal syndrome by reducing mitochondrial oxidative stress.

Oxidative stress–induced cardiorenal syndrome, also referred to as uremic cardiomyopathy, has been extensively studied by multiple research groups over the past two decades. Therefore, the concept itself is not novel. Moreover, the potential effects of NAC in a rat model of CRS have already been examined by the same research team.

 

1. Uremic cardiomyopathy secondary to oxidative stress has been well established for many years, with several important review articles (including those published in Nature Reviews) and original studies on this topic. These references should be cited in the Introduction to provide proper context.

2. In a recent publication from the same group, rats subjected to 5/6 PNX were treated with NAC continuously for 45 days. Given those findings, it is difficult to accept that a single pre-treatment dose of NAC in the PNX model would have a significant effect. The rationale for using a pre-treatment rather than a post-treatment model should be clearly explained.

3. In Figure 5 and the accompanying echocardiography table, the authors state that PNX rats developed reduced EF. However, this is not supported by the representative images, as the M-mode tracing from PNX rats appears to show elevated or at least preserved EF compared to Sham. Furthermore, in their previous publication, rats at 60 days post-PNX did not exhibit reduced EF. Additional explanation is needed to clarify this discrepancy.

4. In Figure 5, the representative NAC-PNX echocardiographic image appears to have mislabeled LVAW and LVPW compared with the PNX image. Additionally, these measurements should be performed during diastole, not systole.

5. In Figure 9, the label “RENAK” is unclear—please clarify its meaning.

6. Figures 14 and 15 would be more appropriate as supplementary figures.

Author Response

Reviewer 2

The aim of this manuscript is to investigate the cardiac and renal protective effects of NAC in a rat model of CKD (5/6 PNX). The authors report that NAC pre-treatment in the 5/6 PNX model attenuates cardiorenal syndrome by reducing mitochondrial oxidative stress.

Oxidative stress–induced cardiorenal syndrome, also referred to as uremic cardiomyopathy, has been extensively studied by multiple research groups over the past two decades. Therefore, the concept itself is not novel. Moreover, the potential effects of NAC in a rat model of CRS have already been examined by the same research team.

1. Uremic cardiomyopathy secondary to oxidative stress has been well established for many years, with several important review articles (including those published in Nature Reviews) and original studies on this topic. These references should be cited in the Introduction to provide proper context.

R=  Many thanks for your comments, to highlight the relevance of uremic cardiomyopathy and mitochondrial impairment, the next paragraph was added to introduction section “In this way, uremic cardiomyopathy and its effects in mitochondrial impairment has been widely studied in recent years, both uric acid and uremic toxins may affect heart mitochondrial respiration and the impairment in metabolism of this organelle also favors the production of these molecules [1]. Uremic toxins like indoxyl sulfate (IS) promote inflammation, oxidative stress and cardiomyocytes apoptosis favoring acute cardiac dysfunction in after renal injury [2] [3]. Meanwhile in patients and in experimental models IS has been related with left ventricular hypertrophy [4].Likewise, uremic toxins has been related with the  SIRT1/3 -PGC-1α axis downregulation in heart  which results in oxidative stress promotion [5–8].”

1. In a recent publication from the same group, rats subjected to 5/6 PNX were treated with NAC continuously for 45 days. Given those findings, it is difficult to accept that a single pre-treatment dose of NAC in the PNX model would have a significant effect. The rationale for using a pre-treatment rather than a post-treatment model should be clearly explained.

R= Many thanks for your comments. As the reviewer mentioned, we recently demonstrated that sustained post-administration of NAC, after  5/6 nephrectomy partially reversed the CRS development[9], highlighting the mitochondrial dysfunction role in the heart damage progression. Furthermore, several studies showed that renal mitochondrial impairment is an early event that precedes the development of others pathophysiological processes classically associated with the CKD development [10–14]. Furthermore, in cardiorenal syndrome type 3, alterations in heart mitochondrial function and structure were reported as early as 48 h after the induction of renal damage[15]. However, in 5/6Nx, it was not yet known at what time the first alterations in cardiac mitochondrial respiration appeared. Therefore, the objectives of this study were focused first on finding the earliest time at which mitochondrial respiration alteration occurred in heart (for which we evaluated at times prior to 10 days; data not shown), with the first alterations being found at 10 days. Once this time was determined, the next goal was to evaluate if from that moment the NAC administration could start to protect the heart mitochondrial function that would be related to the later CRS development. Although, as the reviewer comments, for this beneficial effect to be significantly maintained, it would be necessary to implement a subsequent sustained post-administration regimen like the one used in our previous article [9]. One of the achievements of this work is to highlight that, from the first administration, NAC already exhibits protective effects on cardiac mitochondrial function, which can be associated with cardiac protective effects. Therefore, we are currently conducting for future work a time-based evaluation, including periods between 10 and 45 days, to assess the progression of mitochondrial damage and the effect of sustained NAC administration in combination with the early administration.

1. In Figure 5 and the accompanying echocardiography table, the authors state that PNX rats developed reduced EF. However, this is not supported by the representative images, as the M-mode tracing from PNX rats appears to show elevated or at least preserved EF compared to Sham. Furthermore, in their previous publication, rats at 60 days post-PNX did not exhibit reduced EF. Additional explanation is needed to clarify this discrepancy.

R= The echocardiographic images from each experimental group were reviewed in detail, and the most representative image was added according to the reviewer's comments. The measurements were reviewed; we noted that EF in the group PNX  have high variability compared to the Sham group, in some cases the EF was conserved (PNX 78% versus Sham 84%), although in other case the FE was 55% in PNX, which would explain the diminished of EF in this group. Regarding the evaluation of cardiac function in the publication indicated by the reviewer, we must consider that:

1. a) We know that the EF of the post-PNX group did not change after 60 days may be due to the activation of a compensatory mechanism, as observed in the increase in cardiac fibrosis. Although not in the context of PNX and to support this premise, it has been shown that in aged rats, EF is also not affected compared to adult rats, but there are significant changes in tissue fibrosis processes, corroborating the involvement of compensatory mechanisms [16].
2. b) In the study mentioned by the reviewer, the rats were anesthetized with pentobarbital (PB) (1.9 mg/100 g body weight), while in this manuscript, the rats were subjected to inhaled isoflurane (induction 5% and maintenance at 2.5-3%). It is well known that the use of anesthetics during echocardiographic studies is associated with alterations in functional parameters. For example, in the Fisher 344 rat strain, pentobarbital significantly increases EF, while with isoflurane it is lower, and LV end-diastolic volume is smaller with the use of PB compared to isoflurane [17].

1. In Figure 5, the representative NAC-PNX echocardiographic image appears to have mislabeled LVAW and LVPW compared with the PNX image. Additionally, these measurements should be performed during diastole, not systole.

R= Thank you for your careful and timely comment. We modified the labeling in Figure 5 and added B-mode images during diastole, when cardiac function was measured. In addition, we retained the echocardiographic data for this moment in the Table 2.

1. In Figure 9, the label “RENAK” is unclear—please clarify its meaning.

R= Thank you very much “Renak” was changed to “Renal”

1. Figures 14 and 15 would be more appropriate as supplementary figures.

R= Figures 14 and 15 were moved to supplementary material as supplementary figures 1 and 2

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Reviewer 3 Report

The study of Peralta-Buendía and collegues investigates whether pre-treatment with N-acetylcysteine (NAC) could affect mitochondrial dysfunction in the heart and kidneys in a surgical rat model of kidney fibrosis, subtotal nephrectomy.
The authors provide a significant amount of data, showing that a single NAC administration prior to the surgery protects the cardiac and renal mitochondria, reduced systemic inflammatory response and reduced kidney damage. The study is overall well designed and the results are interesting. I have some concerns that should be answered by the authors during revision, including methodology issues, graphical data presentation and statistical analysis.

Methods section:
1) Lines 81-116: please provide catalogue numbers for each reagent used.

2) The time window used in the study seems rather short. Subtotal nephrectomy (5/6 Nx) induces a slow renal deterioration due to nephron loss, first affecting renal function significantly 3-4 weeks after surgery, and glomerulosclerosis/fibrosis appears from week 5 or 6 after surgery (SNX histology is usually evaluated between weeks 6 to 8 after surgery). Thus, only minimal changes can be expected at 10 days post surgery. Also, apart of a single dose NAC pre-treatment, a post-surgery NAC administration should be studied, to test it as a potential treatment option.

3) Line 173: The Lowry method is suboptimal for protein measurement in samples containing RIPA buffer, as detergents interfere with the assay. Why the authors chose this imprecise method instead of the widely used BCA assay that yields more consistent results?

4) Immunoblots: Data of primary and secondary antibodies are missing (provider, catalogue number, dilution).

5) Original blots:
- In each blot, molecular weight marker should be clearly annotated!
- Plasma troponin and corresponding Ponceau blots do not overlap, and the 40kD troponin band appears at around 30kD accoring to the molecular weight marker;
- Plasma IL-6 blot claims to be IL1B at 21kD, but bands are between 25kD and 37kD;
- Original kidney blots of mitochondrial markeres are missing!
- Original cardiac blots of mitochondrial markeres are missing!
- NRF2-M8 shows a band over 100kD, not 80kD (specificity issues?)
- ACTINA-M8 should be ACTIN-M8
- NRF2 M34 shows a band over 100kD, not 80kD (specificity issues?)
- M25 PGC1a is oversaturated, basically impossible to evaluate!
- M28 PGC1b has 2 stars marking, without explanation. Instead of the company mol.wt. marker photo, annotate your blot's mol.wt marker!
- Blot M45: IL1b seems rather a faint Actin, the 2 bands overlap 100% (!) Please add a well with recombiniant IL1b as positive control.
- TNF original blots are missing.

6) Statistics: ANOVA is inappropriate for analysis of non-Gaussian data (eg. protein expressions). Please re-calculate with Kruskal-Wallis test.

7) Why did the authors choose western blot for troponin, IL1 and IL6 protein determination when much more sensitive and reliable ELISA kits are available? Besides, normalization to Ponceau-S is not a precise method.

Results section:
8) Bar charts are unacceptable, they should be changed to scatter plots in order to show individual values.

9) Each results subheading contains many explanatory sentences that belong to discussion, not the results. Please revise.

10) Figure 2: scale bar should be larger, the present one is basically invisible. The photomicrographs have too low resolution. H&E stained NX kidney shows several fixation artefacts (compared to the Masson stained NX, which does not), this should be changed to a representative sample. The H&E stained NAC kidney photo shows also fixation artefact (shrinking). In Masson stained slides, fibrotic area should appear blue, but this is not present in the NX sample where the arrow is pointing. Please revise.

11) Figure 3: Why are inflammatory markers not measured in the kidney, only in the heart? IL1, TNF, IL6 levels should be provided in kidney samples. Figure 3D: what does "Plamatic" mean?

12) Figure 4: There is no fibrosis visible on any of the heart photomicrographs, please revise. Scale bar issue is same as for kidney samples. Also, please provide similar cutting angle for each heart sample picture otherwise it is impossible to properly compare histology. Also, color of NX H&E heart is totally different from the outher groups, and does not contain the scale bar (also seems a bit magnified comparing nuclear size).

13) Figure 9: "Renak" mitochondria (?)

14) Mitochondrial damage could be further investigated by the estimation of fusion and fission. Did the authors evaluate them?

15) Figure 13: Magnification should be the same for all photos, please correct.

Author Response

Reviewer 3

The study of Peralta-Buendía and colleagues investigates whether pre-treatment with N-acetylcysteine (NAC) could affect mitochondrial dysfunction in the heart and kidneys in a surgical rat model of kidney fibrosis, subtotal nephrectomy.
The authors provide a significant amount of data, showing that a single NAC administration prior to the surgery protects the cardiac and renal mitochondria, reduced systemic inflammatory response and reduced kidney damage. The study is overall well designed, and the results are interesting. I have some concerns that should be answered by the authors during revision, including methodology issues, graphical data presentation and statistical analysis.

• Lines 81-116: please provide catalogue numbers for each reagent used.

R= The catalog numbers and commercial company of each reagent, antibody, and substrate were provided in the supplemental material in Table 1.

2) The time window used in the study seems rather short. Subtotal nephrectomy (5/6 Nx) induces a slow renal deterioration due to nephron loss, first affecting renal function significantly 3-4 weeks after surgery, and glomerulosclerosis/fibrosis appears from week 5 or 6 after surgery (SNX histology is usually evaluated between weeks 6 to 8 after surgery). Thus, only minimal changes can be expected at 10 days post surgery. Also, apart of a single dose NAC pre-treatment, a post-surgery NAC administration should be studied, to test it as a potential treatment option.

R= Thaks for the reviewer comments, as the reviewer comments 5/6Nx nephrectomy is characterized by a sustained decrease over time in kidney function, which leads to the development of CKD. Although some studies have been focused on periods from 28 days onwards, CKD has been fully established. Many others has showed that renal hemodynamic alterations appear from the first moments after the renal mass loss [1,2]. Furthermore, in the first weeks there is an increase in single nephron glomerular filtration rate (GFR), hypertrophy and renal bio-macromolecular synthesis rise  [3–6]. During the first days after surgery, the elevated snGFR leads to higher solute reabsorption witch together with the biosynthetic rise generates an excessive energy demand in the nephron inducing stress in mitochondria [3,7–10]. In fact, we previously showed that mitochondrial ATP production capacity in renal tissues is reduced from 24 after nephrectomy, this together with a pro-oxidant environment are maintained even after 28 days, favoring pathological processes that favor CKD development [7,11,12]. Likewise, similar mitochondrial impaired has been reported other CKD models  like the induced by folic acid and unilateral obstruction of the ureter, meanwhile prevention these early mitochondrial impairment reduces or reverses the development of this pathology[13–15], highlighting the mitochondria role  in pathophysiology of this disease. Considering this background, several authors and we have hypothesized that in CRS type 3 and 4, heart mitochondrial alterations could also be an early event that precedes and triggers other pathological processes like fibrosis and inflammation that lead to heart deterioration. Thus, this study was first focused on finding the earliest time at which mitochondrial respiration alteration occurred in heart after surgery, with the first alterations being found at 10 days. Once this time was determined, the next goal was to evaluate if from that moment the NAC administration could start to protect the heart mitochondrial. Although, as the reviewer comments, for this beneficial effect to be significantly maintained a being consider as potential treatment option, it would be necessary to implement a subsequent sustained post-administration scheme like the one used in our previous works [16].

3) Line 173: The Lowry method is suboptimal for protein measurement in samples containing RIPA buffer, as detergents interfere with the assay. Why the authors chose this imprecise method instead of the widely used BCA assay that yields more consistent results?

R= Thank you for your comments, as the reviewer mentions cationic detergents at high concentration may interference with the Lowry assay by the generation of yellow precipitates. However, it was also reported that the addition of 0.5% SDS w/v (as used in this determination), an anionic detergent, that prevents the formation of such precipitates, allowing overcome this disadvantage even without the need for centrifugation the samples [17]. This allows the use of this technique for the required quantification, likewise we try to use the same technique that we employed in tests with absence of cationic detergent, reducing variations and costs.

4) Immunoblots: Data of primary and secondary antibodies are missing (provider, catalogue number, dilution).

R= The catalog numbers, commercial company and dilution were provided in supplementary Table 1.

5) Original blots:

- In each blot, molecular weight marker should be clearly annotated!

R= The molecular weight ladder used in each membrane was indicated by arrows on the side of the membrane.

- Plasma troponin and corresponding Ponceau blots do not overlap, and the 40kD troponin band appears at around 30kD according to the molecular weight marker.

R= Cardiac troponin was repeated, the original images of plasma troponin and its corresponding loading control were corrected and added in the file with the original images of western blot, indicating the corresponding bands.

- Plasma IL-6 blot claims to be IL1B at 21kD, but bands are between 25kD and 37kD.

R= The IL-6 image was corrected indicating the corresponding bands in the file

- Original kidney blots of mitochondrial markers are missing!

R= The evaluation of mitochondrial mass markers (the subunits of the electron transfer system and ATP synthase complexes) was performed with the OxPhos Rodent WB Antibody Cocktail, which is composed of 5 antibodies. The image corresponding to the kidney mitochondrial marker evaluation was provided with the OXPHOS label in the kidney section and the observed bands refer to each antibody of the cocktail subunits.

- Original cardiac blots of mitochondrial markers are missing!

R= Like in the previous case the image corresponding to the heart mitochondrial marker evaluation was provided with the OXPHOS label in the heart section and the observed bands refer to each antibody of the cocktail subunits.

- NRF2-M8 shows a band over 100kD, not 80kD (specificity issues?)

R= Thanks for the observation, the error in the molecular weight ladder was corrected in the NRF2-M8 image, where it is observed that NRF2 is below the 100 KD band.

- ACTINA-M8 should be ACTIN-M8

R= The membrane title was corrected

- NRF2 M34 shows a band over 100kD, not 80kD (specificity issues?)

R= Like in previous comments the error in the molecular weight ladder was corrected in the NRF2-M34 image.

- M25 PGC1a is oversaturated, basically impossible to evaluate!

R= Many thanks for your comment, PGC1 alpha in renal tissue was remade. The new original images of WB are presented the original WB presentation, this new membrana was used to updated the densitometry analysis and statistical analysis are shown in Supplementary Figure 1.

- M28 PGC1b has 2 stars marking, without explanation. Instead of the company mol.wt. marker photo, annotate your blot's mol.wt marker!

R= The molecular weight ladder used in the membrane was added.

- Blot M45: IL1b seems rather a faint Actin, the 2 bands overlap 100% (!) Please add a well with recombinant IL1b as positive control.

R= To validate our results a new membrane of IL-1 beta was made, using GAPDH as loading control, founding similar behavior. The corresponding new membranes are attached to the modified version of the original WB images. Due to the difficulty in obtaining recombinant IL-1 beta on the days assigned to respond to the reviewers, the data were also validated by evaluating IL-beta levels in the heart using Elisa commercial kit (See Supplementary Fig 4, B).

- TNF original blots are missing.

R= TNF alpha original image was placed in the images section of the heart (M46), along with its corresponding load control. Likewise, the data was validated by commercial ELISA kit as is shown in supplementary Figure 4C, showing the same behavior.

 

6) Statistics: ANOVA is inappropriate for analysis of non-Gaussian data (eg. protein expressions). Please re-calculate with Kruskal-Wallis test.

R= The analysis of protein levels performed by Western Blot was redone using the Kruskal-Wallis test

7) Why did the authors choose western blot for troponin, IL1 and IL6 protein determination when much more sensitive and reliable ELISA kits are available? Besides, normalization to Ponceau-S is not a precise method.

R= Although the ELISA assay is faster and has good sensitivity, it requires pre-optimized commercial kits to generate this advantage. Similarly, the Western blot assay in blood samples is currently often used to corroborate the results obtained by ELISA in different pathologies, reaffirming the usefulness of the WB[18,19]. Due to the short period of time given us to respond to reviewers’ comments and financial resources, the only the IL-1B results could only be confirmed by ELISA, observing the same pattern of behavior as with the Western blot (Supplementary Fig. 4A), supporting the validity of the results obtained.

Results section:
8) Bar charts are unacceptable, they should be changed to scatter plots in order to show individual values.

R= All the graphs were changed to scatter plots with bars allowing the visualization of individual values

9) Each results subheading contains many explanatory sentences that belong to discussion, not the results. Please revise.

R= The subheading names in sections 3.1 to 3.5 of the results section have been substantially reduced.

10) Figure 2: scale bar should be larger, the present one is basically invisible. The photomicrographs have too low resolution. H&E stained NX kidney shows several fixation artefacts (compared to the Masson stained NX, which does not), this should be changed to a representative sample. The H&E-stained NAC kidney photo shows also fixation artefact (shrinking). In Masson-stained slides, fibrotic area should appear blue, but this is not present in the NX sample where the arrow is pointing. Please revise.

R= Figure 2 was corrected, the resolution in the H&E and Masson stained were improved. To make the scale bar easier to view, it was placed within a white background in larger images with a more visible font. The image was corrected to point with the arrows the fibrotic areas.  We appreciate the reviewer’s observation regarding the possible presence of review artifacts in the histological images presented in our manuscript. However, after a detailed review of the histological sections, we did not identify any visible artifacts or inconsistencies that compromise quality or interpretation. To be certain, we double-checked the images in Figures 2 and 4 and found no anomalies attributable to processing errors. We believe the artifacts noted may be attributable to the handling of low-resolution images and inadequate contrast.

11) Figure 3: Why are inflammatory markers not measured in the kidney, only in the heart? IL1, TNF, IL6 levels should be provided in kidney samples. Figure 3D: what does "Plamatic" mean?

R= Thanks for the reviewer comment.  In Fig. 3D “Plasmatic” was change to “Plasma” levels. It has been widely reported that after nephrectomy, renal damage triggers a sustained increase in the production of proinflammatory cytokines by this organ[20] [21,22]. Although we did not evaluate the levels of cytokines in the kidney in this work, we have previous showed in temporal studies in this model a progressive increase in renal inflammation from 24 h to 28 days [11,12,23], which is consistent with the increase in these cytokines in the kidney reported by several authors [24–27], this cytokines are then released into circulation, as we observed by the increases in plasma levels (Fig. 1). Therefore, the objective of evaluating them in the heart was to determine if these cardiorenal mediators were already elevated in tissue at this time, in see if these cardiorenal mediators could be associated with the observed heart mitochondrial damage.

12) Figure 4: There is no fibrosis visible on any of the heart photomicrographs, please revise. Scale bar issue is same as for kidney samples. Also, please provide similar cutting angle for each heart sample picture otherwise it is impossible to properly compare histology. Also, color of NX H&E heart is totally different from the outher groups and does not contain the scale bar (also seems a bit magnified comparing nuclear size).

R= R= Figure 4 was corrected. The resolution in all images was improved and all of them contain their respective scale bars, similar cutting angles were used in the heart images. NX image was changed. The scale bar was placed within a white background with a more visible font, to easier the visualization.

13) Figure 9: "Renak" mitochondria (?)

R= R= Thank you very much “Renak” was changed to “Renal”

14) Mitochondrial damage could be further investigated by the estimation of fusion and fission. Did the authors evaluate them?

R= As the reviewer comments, the cardiac mitochondrial oxidative stress and the fragmentation observed by electron microscopy suggested a shift of mitochondrial dynamic to fission in heart. Likewise, in previous work we showed that the temporal increase in renal mitochondrial fission promotes mitochondrial mass reduction which correlates with the renal fibrosis and CKD promotion[12]. Furthermore, in CRS type III, we showed that the early reduction in membrane potential and oxidative stress in this organelle also favor the increase of fission proteins in heart mitochondria [28]. Therefore, we are currently carrying out the temporal evaluation (between 10 and 60 days after nephrectomy) of the changes in mitochondrial dynamics and biogenesis in heart, which will be integrated into future work still in development.

15) Figure 13: Magnification should be the same for all photos, please correct.

R= Fig 13 was corrected and the photos used all have the same magnification.

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Round 2

Reviewer 2 Report

The authors addressed all the reviewer's comments. 

N/A

Author Response

We  greatly appreciate all the reviewer's comments, that we consider having been very valuable in improving the manuscript.

Reviewer 3 Report

The authors have improved the manuscript according to several previous comments, but some important concerns were not addressed during the revision, and also new questions arise after the revision.

 

At the end of the discussion and before the conclusion heading there should be a "Study limitations" subchapter, as there are several limitations that need to be clearly shown for the readers.

In the introduction lines 69-77 the authors discuss uremic cardiomyopathy and that uremic toxins affect Sirtuin and PGC1a, as rationale for the present study. 
However, ten days post 5/6 nephrectomy is for sure not a uremic stage, as rats happily would have lived for another 10-15 weeks. Uremia is a terminal stage of renal failure, characterized by severe weight loss, unresponsiveness of the animals. Here, NX animals did not lose weight and the increased BUN and creatinine do not reflect renal failure. In addition, no uremic toxins have been measured from the blood, although very likely none of the uremic toxins would have been significantly elevated 10 days post-Nx.
I would re-phrase this paragraph as the used model does not reflect the proposed mechanism of uremia, therefore it is misleading. Also, the short-term experimental end-point need to be discussed in the "Study limitations" section,

I do not agree with the authors that renal inflammatory markers are 100% reflected by systemic inflammation. Inflammatory response short-term (few days) after surgery is reflected mostly in the blood, but 5/6 Nx also induces liver damage (doi: 10.5812/numonthly.37840) with possible inflammatory response and cytokines synthesized in the liver appear sooner in the blood as compared to renal cytokines. A qPCR for TNF, IL1b and IL6 could be performed in a few days from RNA isolation to the results. Otherwise this issue should be mentioned as study limitation.

Regarding the original immunoblot supplementary file, I found many issues. For instance, the original histology photos in the supplementary file are different from the ones in the paper.

Also, spanish names were not revised (eg. "Sirtuina", "Actina"). The molecular weight marker size annotation is only provided in blots of pages 45 to 62, but totally missing in plasma blots (pages 3-9) and cardiac blots (pages 12-41). Please correct these issues for reproducibility.

Also, please comment why is the 120 kD sirtuin-1 band at 75kD in the blot of page 47? Is it possible you quantified an unspecific band? (multiple faded lower bands are also present)

Author Response

Reviewer 3

The authors have improved the manuscript according to several previous comments, but some important concerns were not addressed during the revision, and also new questions arise after the revision.

Detailed comments

1. At the end of the discussion and before the conclusion heading there should be a "Study limitations" subchapter, as there are several limitations that need to be clearly shown for the readers.

R= A subchapter briefly discussing the limitations of the present article was included in the new version of the manuscript before the conclusions subchapter.

 

2. In the introduction lines 69-77 the authors discuss uremic cardiomyopathy and that uremic toxins affect Sirtuin and PGC1a, as rationale for the present study. 
   However, ten days post 5/6 nephrectomy is for sure not a uremic stage, as rats happily would have lived for another 10-15 weeks. Uremia is a terminal stage of renal failure, characterized by severe weight loss, unresponsiveness of the animals. Here, NX animals did not lose weight and the increased BUN and creatinine do not reflect renal failure. In addition, no uremic toxins have been measured from the blood, although very likely none of the uremic toxins would have been significantly elevated 10 days post-Nx.
   I would re-phrase this paragraph as the used model does not reflect the proposed mechanism of uremia, therefore it is misleading. Also, the short-term experimental end-point need to be discussed in the "Study limitations" section.

 

R= We agree, 10 days after nephrectomy, a uremic stage has not been yet developed. In this stage glomerular filtration rate per nephrectomy and renal hemodynamic parameters has been altered[1–6], however, as the reviewer mentions, it is very unlikely that uremic toxins are been elevated. However, we were previously requested by other reviewers to include in the introduction chapter, the relationship about uremic cardiomyopathy with the mitochondrial biogenesis changes. Thus, the mentioned paragraphs were re-paraphrased to emphasize that the present study is not focused on proposing uremia as one of the early mechanisms leading to cardiac mitochondrial impairment short-term experimental end-point need, although other studies have shown that the elevation of uremic toxins can inhibit mitochondrial biogenesis in cardiomyocytes, they were carried out in more advanced stages. This point was also discussed in Study limitation section.

 

3. I do not agree with the authors that renal inflammatory markers are 100% reflected by systemic inflammation. Inflammatory response short-term (few days) after surgery is reflected mostly in the blood, but 5/6 Nx also induces liver damage (doi: 10.5812/numonthly.37840) with possible inflammatory response and cytokines synthesized in the liver appear sooner in the blood as compared to renal cytokines. A qPCR for TNF, IL1b and IL6 could be performed in a few days from RNA isolation to the results. Otherwise, this issue should be mentioned as study limitation.

R= We respect the reviewer's point of view. As the reviewer comments, the 5/6NX also induce early liver impairment, like dyslipidemia, mitochondrial damage, inflammation and apoptosis that may promote liver cytokines synthesis. However, our previous studies have shown that these changes (hyperlipidemia, inflammation, oxidative stress, and apoptosis) appear in the liver as early as 2 weeks after surgery [7], a later time than the experimental end-point of this study. Although we do not rule out the possible partial participation of the liver in systemic inflammation, we believe that we have demonstrated the increase in renal and bloodstream inflammation markers by various techniques, which are consistent with what has been reported in the literature[8]. Although for these reasons we do not consider qPCR to be essential, this was also discussed in the Study limitation section.

 

 

4. Regarding the original immunoblot supplementary file, I found many issues. For instance, the original histology photos in the supplementary file are different from the ones in the paper.

R= The corresponding images used in histology and electron microscopy now appear at the end of the supplementary material and non-publishable material.

 

5. Also, spanish names were not revised (eg. "Sirtuina", "Actina"). The molecular weight marker size annotation is only provided in blots of pages 45 to 62 but totally missing in plasma blots (pages 3-9) and cardiac blots (pages 12-41). Please correct these issues for reproducibility.

R= The molecular weight markers size is placed on the left of each WB image and the reference to the commercial protein carrier used was placed on each image. The Spanish name was change to English

 

6. Also, please comment why is the 120 kD sirtuin-1 band at 75kD in the blot of page 47? Is it possible you quantified an unspecific band? (multiple faded lower bands are also present)

R= Interestingly, as the reviewer comment, Sirt1 WB evaluation in kidney showed a band around 75 KDa, this shorter form of SIRT1 has been previously reported as a result of proteolytic cleave of the full-length form of SIRT1 (120kDa) by proteases, giving rise to an N-terminal form[9,10], which is even reported in the Uniprot database (Uniprot Q96EB6). Interestingly, other commercial antibodies are reported to be able to identify this cleaved form of SIRT1 (Abcam ab189494 Anti-SIRT1, Proteintech  SIRT1 Monoclonal, 60303-1-Ig, Human Sirtuin 1/SIRT1 Antibody MAB7714, etc). Therefore, to specify that the band being detected corresponds to the 75kDa form, the original image in the supplementary material and the Fig. 11 in the manuscript were modified with the legend SIRT1 75KDa fragment, specifying this weight. Because we still do not know the biological implication of this shorter form in the model, this was discussed in the Study limitations section.

 

Bilbliography

[1]           T.H. Hostetter, J.L. Olson, H.G. Renke, M.A. Venkatachalam, B.M. Brenner, Hyperfiltration in remnant nephrons: a potentially adverse response to renal ablation, J. Am. Soc. Nephrol. 12 (2001) 1315–25. https://doi.org/10.1681/ASN.2009020171.

[2]           B.M. Brenner, Nephron adaptation to renal injury or ablation., Am. J. Physiol. 249 (1985) F324–F337. https://doi.org/10.1152/ajprenal.1985.249.3.F324.

[3]           M.W. Taal, B.M. Brenner, Adaptation to Nephron Loss and Mechanisms of Progression in Chronic Kidney Disease, in: K. Skorecki, G.M. Chertow, P.A. Marsden, M.W. Taal, A.S.L. Yu (Eds.), Brenner & Rector’s The Kidney, Ninth Edit, Elsevier, 2012: pp. 1918–1971. https://doi.org/10.1016/B978-1-4160-6193-9.10051-X.

[4]           I. Sinuani, Z. Averbukh, I. Gitelman, M.J. Rapoport, J. Sandbank, M. Albeck, B. Sredni, J. Weissgarten, Mesangial cells initiate compensatory renal tubular hypertrophy via IL-10-induced TGF-beta secretion: effect of the immunomodulator AS101 on this process., Am. J. Physiol. Renal Physiol. 291 (2006) F384-94. https://doi.org/10.1152/ajprenal.00418.2005.

[5]           P. Hauser, A. Kainz, P. Perco, H. Bergmeister, C. Mitterbauer, C. Schwarz, H.M. Regele, B. Mayer, T.W. Meyer, R. Oberbauer, Transcriptional response in the unaffected kidney after contralateral hydronephrosis or nephrectomy, Kidney Int. 68 (2005) 2497–2507. https://doi.org/10.1111/j.1523-1755.2005.00725.x.

[6]           G. Wolf, F.N. Ziyadeh, Molecular mechanisms of diabetic renal hypertrophy, Kidney Int. 56 (1999) 393–405. https://doi.org/10.1046/j.1523-1755.1999.00590.x.

[7]           Z.A. Ceja-Galicia, F.E. García-Arroyo, O.E. Aparicio-Trejo, M. El-Hafidi, G. Gonzaga-Sánchez, J.C. León-Contreras, R. Hernández-Pando, M. Guevara-Cruz, A.R. Tovar, P. Rojas-Morales, A.K. Aranda-Rivera, L.G. Sánchez-Lozada, E. Tapia, J. Pedraza-Chaverri, Therapeutic Effect of Curcumin on 5/6Nx Hypertriglyceridemia: Association with the Improvement of Renal Mitochondrial β-Oxidation and Lipid Metabolism in Kidney and Liver, Antioxidants. 11 (2022) 2195. https://doi.org/10.3390/antiox11112195.

[8]           I. Amador-Martínez, O.E. Aparicio-Trejo, B. Bernabe-Yepes, A.K. Aranda-Rivera, A. Cruz-Gregorio, L.G. Sánchez-Lozada, J. Pedraza-Chaverri, E. Tapia, Mitochondrial Impairment: A Link for Inflammatory Responses Activation in the Cardiorenal Syndrome Type 4, Int. J. Mol. Sci. 24 (2023). https://doi.org/10.3390/ijms242115875.

[9]           H. Oppenheimer, O. Gabay, H. Meir, A. Haze, L. Kandel, M. Liebergall, V. Gagarina, E.J. Lee, M. Dvir-Ginzberg, 75-kd sirtuin 1 blocks tumor necrosis factor α-mediated apoptosis in human  osteoarthritic chondrocytes., Arthritis Rheum. 64 (2012) 718–728. https://doi.org/10.1002/art.33407.

[10]        A. Kumar, Y. Daitsh, L. Ben-Aderet, O. Qiq, J. Elayyan, G. Batshon, E. Reich, Y.H. Maatuf, S. Engel, M. Dvir-Ginzberg, A predicted unstructured C-terminal loop domain in SIRT1 is required for  cathepsin B cleavage., J. Cell Sci. 131 (2018). https://doi.org/10.1242/jcs.214973.

 

 

Author Response File: Author Response.pdf