Myocardial Remodeling in Early Chronic Kidney Disease—Mineral and Bone Disorder Model with Low Bone Turnover

Round 1

Reviewer 1 Report

This is an area of potentially important clinical research. The study is well designed and presented. 

Main concerns:

1)    The bone disease reported here is low bone turnover; the question is whether this is also the case in humans with very early CKD or is this just something related to their particular animal model being studied here? This point  needs to be addressed based on information from the literature.

2)    Data on early myocardial remodeling is very interesting and detailed, but the authors should spend more time in the discussion delineating potential mechanisms. They seem to believe it is not related to FGF23, for example, which is described as a very early biochemical abnormality in human CKD. It would be difficult to assure the FGF23 levels they found had no role unless using some other experimental approaches such as testing the effect of FGF23 inhibitors compounds.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

The reviewer's comments are listed below.

1.　 Did the CKD-MBD model show any signs of inflammation? When myocardial inflammation leads to the loss of cardiomyocytes, fibrosis occurs to fill the resulting void, and compensatory fibrosis is initiated to maintain the structural integrity of myocardial tissue. Myofibroblasts expressing α-smooth muscle actin play a pivotal role in this pathological fibrotic process, distinct from normal fibroblasts. Furthermore, as myocardial fibrosis progresses, the stiffness of myocardial tissue increases, leading to impaired left ventricular diastolic function. Angiotensin II and endothelin act as triggers for myocardial fibrosis, while growth factors such as TGF-β, matrix metalloproteinases (MMPs) involved in collagen degradation, tissue inhibitors of MMPs (TIMPs), and pro-inflammatory cytokines like TNF-α are known to participate in this process. Have you identified such markers in your study? Additionally, have you observed the presence of KLF5, which plays a significant role in cardiac hypertrophy and organ fibrosis, as another marker?

 

2.　 Among the factors associated with CKD-MBD, fibroblast growth factor 23 (FGF23) is the most closely related to the prognosis. It is secreted by bone cells, and when it binds to the complex of Klotho and FGF receptors, it inhibits the expression of the sodium-phosphate cotransporter (NaPi-II) in the proximal tubules, leading to reduced phosphate reabsorption. Klotho expression begins to decrease in the early stages of CKD, but its renal protective effects have been confirmed through animal experiments. FGF23 is also known to increase from the early stages of CKD. As a poor prognostic factor, FGF23 itself has been implicated in causing left ventricular hypertrophy.　In authors study, why was there no significant increase in FGF23 levels? Please provide the reasons behind this absence of notable elevation in FGF23 levels.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have developed a rat model of mild CKD, where they study the bone and heart phenotype in the absence of massive changes in phosphate metabolism. Their findings that cardiac fibrosis precedes hypertrophy and that the myocardium accumulates phosphate is interesting and important to report. However, there are some questions and confusions about the model and the data interpretation.

 

Serum levels of intact FGF23 are elevated in Nx2 and Nx6 rats, and serum phosphate is increased in Nx6 rats, as shown in Table S3. Therefore, the authors’ statement that they study phenotypic changes that occur in a CKD-MBD model without increases in circulating factors associated with CKD is incorrect. In CKD patients FGF23 levels rise early, but phosphate very late, so the Nx6 rats cannot be described as a model for mild/early CKD. This needs to be corrected, and the interpretation of all findings in relation to CKD stages in patients need to be modified.

 

The accumulation of phosphate in the myocardium, and that it occurs before increases in serum phosphate levels, is interesting, as it indicates that determining serum phosphate levels might not really reflect tissue phosphate levels, and that the accumulation of phosphate in tissues might be harmful, and as shown here associates with cardiac fibrosis. The authors should discuss this in more detail. They should also conduct van Kossa stainings of heart sections to determine if they can detect calcifications in their CKD rats. It could be that phosphate is indeed intracellular, as speculated by the authors. However, CKD is associated with soft tissue calcifications, which has been mainly studied in the vasculature but barely in the heart. It would be important to know if in the authors’ rat model the myocardium is calcified, which would indicate that such calcification events occur in the absence of elevations in serum phosphate levels.

 

The authors need to tone down the interpretation of their signaling data. Analyzing the expression levels of signal mediators, such as calcineurin or ERK, does not provide any insights into their level of activity. Studying activation states of these mediators would require protein analyses (e.g. analyze the phosphorylation of mediators by Western blotting).

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed satisfactorily all my queries.

Reviewer 2 Report

I am satisfied with the revisions that have been made by the authors.

Reviewer 3 Report

The authors have addressed my concerns and comments by editing the manuscript. I have no further questions.