Changes in Skeletal Muscle Atrophy over Time in a Rat Model of Adenine-Induced Chronic Kidney Disease

Please, find my comments next to the answers provided by the authors

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Reviewer 1, Comments 1: The adenine-induced chronic kidney disease model in rodents (both mouse and rat) is well established, however the authors should provide evidence (in a supplementary figure) that the disease actually occurred after 4 weeks of 0.75% adenine diet. The authors in the discussion said the study lacks this information and referred to a previously published paper. Histological analysis of kidney, the measurement of the serum creatinine levels, the simplest evaluation of the weight of the treated rats are useful to verify the presence and stage of the disease. None of these analyses have been reported. Another well-established marker that can be evaluated, by the way involved in muscle atrophy induction, is the blood levels of the soluble pro-cachectic factor activin A.

Reviewer 1. Response 1: Thank you for your comment. In our previous study, we have demonstrated 1) the histological analysis of kidney, 2) the measurement of the serum creatinine levels, and 3) the weight of the treated rats of this experimental protocol. We have reported these results in the published paper [Ref# 37]. The study has demonstrated that 1) the enlargement of urinary cavities on the H&E-stained sections and fibrosis of the renal interstitium on the Elastica-Masson-stained sections in CKD rats at 20 weeks, 2) in the CKD rats, BUN, CRE, IP, and IPTH peaked at 12 weeks and decreased significantly at 16 and 20

weeks, except I-PTH, and the BUN, CRE, IP (except at 16 weeks), and I-PTH levels were significantly higher in the CKD rats than in the sham rats from 12 to 20 weeks, and 3) the body weights of CKD rats were significantly lower than of sham rats from 12 to 20 week. We have already confirmed and reported the evidence of CKD condition by the 4 weeks of 0.75% adenine diet. Therefore, we are not able to include these results in current study. Additionally, we recognize the significance of the soluble pro-cachectic factor activin A as a marker of muscle atrophy induction. Given these limitations, we will provide a more comprehensive validation of CKD model.

I am well aware that the authors already proven the development of the CKD in rats as the consequence of the 0.75% adenine diet. The papers describing the analyses are cited in the manuscript. However, exactly because the disease is induced by the diet and is not a genetic disorder, it is mandatory for the authors to demonstrate that rats present the sign of the disease. This means that for each animal used in the different experiments, authors must provide at least one evidence of the disease. Only in this way, it is possible to correlate the atrophy status with the CKD.

Reviewer 1. Comments 2: The number of rats in each group (control; adenine treated; different time points) must be indicated.

Reviewer 1. Response 2: Thank you for the comment. We have added the number of rats in each group (n = 7 in each group). In the revised manuscript, page 2, lines 77.

Reviewer 1. Comments 3: The authors analysed the level of atrophy at two time points. Immediately after adenine diet withdraw (12 weeks of age) and 8 weeks later (20 weeks of age). At the first time point (12 w) authors evidenced reduced CSA of both type I and II fibres, but only type II are still smaller at 20w. An intermediate time point would be useful in understanding the trend, whereas a later time point would be helpful in evaluating whether the observed modifications are maintained or there are additional changes in the atrophy status.

Reviewer 1. Response 3: Thank you for the comment. We also agree that including an intermediate time point and later time point would provide valuable insight into changes in the atrophy status. However, in conducting this experiment, we decided to narrow down the time points to be evaluated, because the number of rats to be sacrificed will increase if the number of time points to be evaluated is large. Therefore, in this study, we focused on two time points, 12 weeks and 20 weeks, to evaluate changes in muscle atrophy. For these reasons, we ask for your understanding that we are evaluating at two time points. We would like to evaluate changes in muscle atrophy at shorter or longer time points in future studies.

I know very well that ethically there is a need to reduce the number of experimental animals, however their use and number (clearly defined in a statistical way) must be linked to the conclusions that can be drawn. It is not a question of planning future experiments but of understanding whether the data collected in this manuscript are conclusive.

 Reviewer 1. Comments 4: In the figure analyzing the CSA of muscle, the number of fibers analyzed must be reported. In the figure reporting the SDH staining, the number of different fields analyzed must be reported. Furthermore, in addition to the global intensity of staining, also the number of SDH positive fibers in the different conditions have to be reported. It may give interesting information.

Reviewer 1. Response 4: Thank you for your comment. We randomly selected five fields and measured 50 fibers in each field, then the mean CSA for one muscle fiber was calculated. We have mentioned this point in the revised manuscript (page 2, lines 94-95). In addition, we have revised the figure captions to include the number of measured fibers and fields in in each rat (page 4, lines 156-158).

To evaluate mitochondrial activity, we performed the SDH staining section with the previously reported method [Ref #7]. We also agree that reporting the number of SDH-positive fibers in the different condition could provide valuable information. However, we were unable to perform and evaluate the SDH staining under different conditions before the deadline for submission of the revised manuscript. We appreciate your understanding of this situation. We hope to perform future study to evaluate the SDH-positive fibers under another condition of SDH staining.

Counting SDH positive fibers is a long and tedious process, but could certainly have been accomplished in the time required for the review

 Reviewer 1. Comments 5: Why were the back muscles analysed for the CSA and SDH, while TA muscles were used for the gene expression experiments?

Reviewer 1. Response 5: Thank you for your comment. We chose to analyze the back muscles because patients with CKD are likely to develop atrophy of the trunk muscles, which often leads to significant clinical issues such as a kyphosis of lumbar spine. Despite this, there have been no previous reports examining atrophy of the back muscles specifically in CKD models, which motivated our focus on this area. For the gene expression experiments, we based on previous studies that have utilized the TA muscles for the gene expression experiments.

If the authors want to focus on the back muscles due to similarity to CKD patients, gene expression studies should also be conducted on the back muscles and compared with those of the TA.

 Reviewer 1. Comments 6: Other muscles should have been analysed to provide a comprehensive picture of the atrophy status/evolution of the adenine-induced CKD rats.

Reviewer 1. Response 6: Thank you for your comment. We also agree that other muscles should have analyzed for comprehensive understanding of the muscle atrophy in CKD rats. Back muscle of rats has been considered to have much more type I (slow muscle fiber) than type II (fast muscle fiber) muscle fiber, on the other hand, TA muscle of rats contained much more type II (fast muscle fiber) than type I (slow muscle fiber). The evaluation of CSA and mitochondrial activity in the TA or quadriceps muscle, which are type II dominant muscle, and the investigation of muscle related genes expression in the soleus muscle, which is type I dominant muscle, could provide more comprehensive picture of muscle atrophy due to CKD. We have mentioned this point as a limitation of this study in the revised manuscript (page 8, lines 276-287).

Reviewer 1. Comments 7: I always suggest to use for qPCR data normalization more than one housekeeping gene. You well know that no housekeeping gene is completely stable. And G3PDH may undergo some alterations in a condition such as CKD in which metabolic changes are known.

Reviewer 1. Response 7: Thank you for your comment. We acknowledge this limitation in our current study and will incorporating multiple housekeeping genes in future research.

Reviewer 1. Comments 8: In addition to MyoD and myogenin that are markers of satellite cells activation, another transcription factor to test should be myostatin. If myostatin is upregulated, both muscle protein metabolism and satellite cell function should be negatively affected, causing muscle wasting.

Reviewer 1. Response 8: Thank you for your comment. We agree that myostatin is an important transcription factor of muscle wasting. While we did not include myostatin in our current study, we plan to include it in our future research for more comprehensive study.

Performing a qPCR to evaluate myostatin expression is certainly a feasible experiment in the time required for the review.

Reviewer 2. Comments 1: Why did the authors choose these time points and what are their expectations with prolonged time of observation

Reviewer 2. Response 1: Thank you for your comment. Based on our previous study, it was reported that 4-week treatment with the adenine diet from 8 to 12 weeks of age can create a CKD model equivalent to stage 4 at 20 weeks of age. Therefore, we considered it appropriate to compare the time points at 12 weeks, representing early-stage CKD, and 20 weeks, representing late-stage CKD.

In addition, as the response 3 of Reviewer 1, we also agree that including an intermediate time point and later time point would provide valuable insight into changes in the atrophy status. However, in conducting this experiment, we decided to narrow down the time points to be evaluated, because the number of rats to be sacrificed will increase if the number of time points to be evaluated is large. Therefore, in this study, we focused on two time points, 12 weeks and 20 weeks, to evaluate changes in muscle atrophy. For these reasons, we ask for your understanding that we are evaluating at two time points. We would like to evaluate changes in muscle atrophy at shorter or longer time points in future studies.