Thank You for Your Consideration s1

Dear Sir:

Thank you very much for your kind message. We have just finished the revised work of our (Manuscript-ID 107204 - 1) entitled “Serum soluble alpha-Klotho levels in patients under hemodialysis.” Now, we are mailing the revised manuscript to you

Enclosed please find the Revised Version of our manuscript. As suggested by a reviewer, we answered and rewrote like the following answer list for each comment. Although I should add some sentences as their advices, I have made my manuscript shorten (Word Count for: Abstract: 183, Text: 1491) without much speculation as your suggestion. Could you please consider our revised manuscript to publish on “Clinical Nephrology"?

Thank you for your consideration.

Sincerely yours,

Keitaro YOKOYAMA MD.

Division of Kidney and Hypertension, Department of Internal Medicine,

The Jikei University School of Medicine

Answers to comments made by Reviewer 1

1. Distribution of sαKl would be quite relevant for this preliminary paper, because this is the first to examine these levels in ESRD patients. In addition, the authors should examine whether treatment for bone and mineral disorders (such as phosphate binders, active vitamin D, and cinacalcet HCL) is associated with an increase or decrease in the sαKl levels.

As you pointed out, it is not clear whether circulating s-Klotho in dialysis patients is a biologically active molecule. Therefore, we should examine the association with parameters of bone disease or with clinical events. However, in this small number of patients study, we could not found any association between treatment for bone and mineral disorders (such as phosphate binders, active vitamin D) and the sαKl levels. Now we are performing the prospective cohort study to evaluate the change of the sαKl levels after 6 month-treatment with cinacalcet. (P.13, line.5)

2. Page 8: Were blood samples drawn from the arterial line, but not from the arteriovenous fistula?

Thank you for your pointing out my mistake. We rewrote “Blood samples were drawn from the arteriovenous at the start of an HD session after a 2-day interval from the last session.” (P.8, line.12)

3. Page 9: Why did the authors used Spearman's rank correlation coefficient? Were the variables not normally distributed?

Because intact PTH and FGF-23 were not normally distrbuted, we used Spearman's rank correlation test for univariate analysis.

4. P10-11: The additional work for the dialyzance of sαKl should be converted to the Results section.

As your advices, I converted the additional work for the dialyzance of sαKl to the Results section. (P.9, line.4)

5. P11: Is there any evidence that sαKl is released from the parathyroid glands?

As your suggestion, I mentioned as below.

However, there no evidence that sαKl is released from the parathyroid glands, so the change of sαKl after total parathyroidectomy should be measured to clarify this point. (P.12, line.5)

6. P13: Why can the authors attribute the diminished association between FGF23 and sαKl to the accumulation of FGF23? Rather, it should be stated that it is unclear why the negative correlation between FGF23 and sαKl, observed in healthy subjects, was disappeared in dialysis population.

As your suggestion, I mentioned as below.

It is unclear why the negative correlation between FGF23 and sαKl, observed in healthy subjects, was disappeared in dialysis population. However, these results suggest that this negative correlation might be masked because of the accumulation of FGF23 in dialysis patients. (P.12, line.18)

7. The authors should also cite the negative data for the role Na-K ATPase on PTH secretion. (Kidney Int, Maetuseviene et al)

As your advice, I referred Kidney Int, Maetuseviene et al (P.11, line.18)

8. Several abbreviations were used without appropriate explanation, such as “Ca” and “P” in the Introduction.

Thank you for your advices. I rewrote as below.

calcium (Ca) and phosphorus (P) (P.6, line.4)

Fibroblast growth factor 23 (FGF23) (P.6, line.8)

hemodialysis (HD) (P.7, line.4)

9. The authors used both “P” and “Pi” in the manuscript, either of which should be replaced.

As your advice, I used just “P” in the manuscript.

Keitaro YOKOYAMA MD.

Division of Kidney and Hypertension, Department of Internal Medicine,

The Jikei University School of Medicine

Answers to comments made by Reviewer 2

1. The main weaknesses of this study are that no outcome data are reported (association with parameters of bone disease or with clinical events). No data on 1, 25 vit D are reported. Furthermore we still ignore whether circulating s-Klotho in dialysis patients is a biologically active molecule.

It is not clear whether circulating s-Klotho in dialysis patients is a biologically active molecule. Therefore, we should examine the association with parameters of bone disease or with clinical events. However, in this small number of patients study, we could not found any association between treatment for bone and mineral disorders (such as phosphate binders, active vitamin D) and the sαKl levels.

2. Furthermore it is still unknown whether s-Klotho reflects the expression of Klotho at organ level.

As your point, I mentioned as below.

However, there no evidence that sαKl is released from the parathyroid glands, so the change of sαKl after total parathyroidectomy should be measured to clarify this point. (P.12, line.5)

3. All these weaknesses need to be frankly faced in the introduction (which may be better focused and shortened) and in the discussion. The discussion should be factual and should specifically deal with the scope of the study, the limitation of present findings and include a conclusive paragraph on "where we go from here".

Although I should add some sentences as their advices, I have made my manuscript shorten (Word Count for: Abstract: 184, Text: 1491) without much speculation as your suggestion.