Dialysate calcium, alfacalcidol, and clinical outcomes: A post-hoc analysis of the J-DAVID trial.

The selection of dialysate calcium concentration (D-Ca) is still controversial among chronic hemodialysis (HD) regimens. We examined the trajectories of CKD MBD parameters among the J-DAVID trial participants to see the effect of D-Ca and alfacalcidol. The trial was an open-label randomized clinical trial including 976 HD patients with intact PTH of 180 pg/mL or lower which compared the users of vitamin D receptor activator (oral alfacalcidol) and non-users over a median of 4 years. The main D-Ca used at baseline were 3.0 mEq/L in 70% and 2.5 mEq/L in 25%, respectively. The primary endpoint was the composite of fatal and non-fatal cardiovascular events and the secondary endpoint was all-cause mortality. Multivariable Cox proportional hazard regression analyses in which D-Ca was included as a possible effect modifier and serum laboratory data as time-varying covariates showed no significant effect modification for composite cardiovascular events or all-cause mortality. This post hoc analysis showed that the effects of alfacalcidol on cardiovascular outcomes were not significantly modified by D-Ca.

Introduction

The hemodialysis (HD) regimen for kidney failure remained experience-based. They consist of session time and frequency of HD, the material of membrane and size of the dialyzer, vascular access, and dialysate composition. Dialysate composition includes sodium, potassium, glucose, acetate, bicarbonate, and calcium. Among them, dialysate calcium (D-Ca) may play an important role in the management of chronic kidney disease-mineral and bone disorders (CKD-MBD). CKD-MBD is one of the major complications leading to cardiovascular disease (CVD), vascular calcification, bone fracture, and all-cause mortality [1].

Ideally, the dialysis regimen should be individualized [2] as the patient’s status such as age, body size, and co-morbid conditions are variable. However, most dialysis units are using a central dialysis fluid delivery system (CDDS), at least in Japan [3]. Dialysate is selected and fixed in each facility according to the choice of the primary physician. Currently, D-Ca is mostly 2.5 to 3.0 mEq/L in Japan as suggested by KDIGO clinical practice guideline (CPG) based on observational studies [1].

Many observational studies showed that the use of vitamin D receptor activator (VDRA) was associated with a lower risk of all-cause mortality [4, 5], cardiovascular mortality [6], and incident cardiovascular events [7] in hemodialysis patients. In addition, a large cohort study [4] showed the association between VDRA use and all-cause mortality was found regardless of intact parathyroid hormone (intact PTH) levels. VDRAs are shown to have pleiotropic actions which are potentially beneficial in patients with chronic kidney disease [8]. However, in the Japan Dialysis Active Vitamin D (J-DAVID) trial [9], treatment with alfacalcidol did not reduce the risk of composite cardiovascular events or the risk of all-cause mortality in hemodialysis patients without secondary hyperparathyroidism. Then, we hypothesized that dialysate calcium concentration (D-Ca) might have influenced the serum parameters of CKD-MBD and modified the effects of alfacalcidol on these clinical outcomes in the trial. We tested this hypothesis in this post hoc analysis of the J-DAVID trial data.

Methods

We used the data obtained from the participants of the J-DAVID trial [9, 10] to examine the effect of D-Ca on the CKD MBD parameters and outcomes. In brief, during the recruitment period from July 1, 2008, to January 26, 2011, a total of 1,289 patients were assessed for eligibility and 976 patients were randomized to VDRA (oral alfacalcidol) or no VDRA (control). The median follow-up was 4 years, and the all-cause mortality rates were 18.2% in VDRA and 16.8% in control, respectively. During the study period, serial data of mineral metabolism were collected such as corrected Ca, phosphate, and intact PTH every 6 months in addition to baseline and 3 months. Majority of patients (95%) used D-Ca of 2.5 mEq/L or 3.0 mEq/L, and only the small proportion of patients used D-Ca of 2.75 mEq/L or 2.9 mEq/L. Therefore, comparison was done between the two groups of D-Ca of 2.5 and 3.0 mEq/L.

In the original J-DAVID trial, the primary outcome was the composite of fatal and non-fatal cardiovascular events (N = 188), and the secondary outcome was all-cause mortality (N = 169). To increases the power of statistical analysis, this post hoc analysis additionally considered the combined outcome including both the primary and secondary outcomes (N = 277).

To examine the difference in the effect of the alfacalcidol use on the clinical events between the D-Ca on the study enrollment, we conducted multivariable Cox proportional hazard regression analyses considering the time-varying treatment. The regression models included a cross-product term between the treatment and the baseline D-Ca value. The estimators were adjusted for the baseline covariates (sex, age, vintage of dialysis, and history of CVD, diabetic kidney disease, body mass index, C-reactive protein, serum levels of albumin and phosphate, corrected calcium, intact PTH, HDL cholesterol, hemoglobin, systolic blood pressure, and use of intravenous iron) and the time-varying covariates (body mass index, C-reactive protein, serum levels of albumin, phosphate, corrected calcium, intact PTH, hemoglobin, HDL-cholesterol, use of intravenous iron, systolic blood pressure).

The distributions of the covariates on the baseline were shown in the original paper [4]. To convert serum level expressed mg/dL to mmol/L, multiply by 0.25 for serum calcium and 0.323 for serum phosphate.

The J-DAVID trial was conducted in accordance with the principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Studies by the Ministry of Health, Labor and Welfare, Japan (the original 2003 version, which was modified in 2004 and 2006). The protocol and revisions of this trial were approved by the ethics committee at the Osaka City University Graduate School of Medicine in Japan (approval numbers 1227, 1297, 1385, and 1525) and by the relevant ethics committees or institutional review boards at the study sites. All participants gave written informed consent before the study. The protocol of post hoc analyses using the original J-DAVID trail was reviewed and approved by the ethics committee of the Osaka City University Graduate School of Medicine (Number 4420 on September 26, 2019).

Results

Data of predefined laboratory abnormalities is available in eTable 4 in the original paper [9]. In Table 1 and Fig 1, the trends of serum levels of corrected Ca, intact PTH, phosphate, and in both D-Ca 2.5 and 3.0 mEq/L were shown. Although all three variables were within the target ranges in most patients, the intervention group showed a temporal increase in corrected Ca and a decrease in intact PTH after starting alfacalcidol, and these changes returned to the initial levels thereafter. These changes were similarly observed in both D-Ca 2.5 and 3.0 mEq/L users.

Table 1

Prevalence of laboratory abnormalities by allocation and dialysate calcium concentration at each study month.

Abnormalities	Allocation	D-Ca (mE/L)	Baseline	3M	6M	12M	18M	24M	30M	36M	42M	48M
Corrected Calcium > 10.0 mg/dL	Intervention	2.5	1.5%	23.0%	19.2%	12.8%	10.1%	12.1%	9.0%	8.5%	11.2%	11.8%
		3.0	4.5%	31.5%	23.4%	17.1%	13.7%	10.4%	13.6%	10.2%	9.9%	8.2%
	Control	2.5	0.8%	1.7%	2.6%	7.2%	5.5%	4.8%	7.0%	3.1%	2.2%	6.0%
		3.0	5.7%	8.3%	6.9%	8.7%	10.8%	8.5%	11.4%	10.5%	11.3%	6.9%
Corrected Calcium > 11.0 mg/dL	Intervention	2.5	0.0%	1.6%	3.2%	0.9%	0.9%	0.0%	1.0%	3.2%	2.2%	1.2%
		3.0	0.0%	7.2%	3.5%	2.6%	2.4%	1.1%	0.4%	1.2%	0.4%	0.4%
	Control	2.5	0.0%	0.8%	0.0%	0.9%	1.8%	1.0%	1.0%	1.0%	0.0%	0.0%
		3.0	0.0%	0.9%	1.3%	0.3%	0.3%	1.4%	0.4%	0.8%	0.8%	0.9%
Phosphate> 6.0 mg/dL	Intervention	2.5	0.0%	23.8%	20.8%	18.8%	19.3%	15.0%	22.0%	19.1%	12.4%	12.9%
		3.0	0.0%	17.1%	19.9%	23.7%	20.1%	18.6%	21.5%	17.2%	19.8%	22.1%
	Control	2.5	0.0%	17.8%	17.1%	14.4%	14.7%	14.4%	19.0%	21.9%	15.2%	21.4%
		3.0	0.0%	13.2%	11.0%	16.2%	13.6%	18.8%	16.2%	20.7%	19.4%	20.7%
Phosphate> 7.0 mg/dL	Intervention	2.5	0.0%	10.3%	6.4%	4.3%	7.3%	2.8%	6.0%	6.4%	4.5%	4.7%
		3.0	0.0%	5.6%	6.0%	6.2%	5.5%	5.0%	5.7%	5.1%	3.7%	7.4%
	Control	2.5	0.0%	7.6%	6.0%	6.3%	5.5%	1.9%	9.0%	6.2%	5.4%	7.1%
		3.0	0.0%	1.5%	1.3%	6.2%	4.4%	6.4%	3.3%	5.5%	4.0%	5.2%
Intact PTH> 240 pg/mL	Intervention	2.5	0.0%	1.6%	0.8%	2.7%	0.9%	9.4%	6.1%	11.0%	12.6%	14.6%
		3.0	0.0%	0.4%	2.2%	1.9%	5.3%	2.0%	6.2%	9.1%	10.9%	15.3%
	Control	2.5	0.0%	4.4%	6.3%	13.6%	17.3%	22.5%	15.6%	13.0%	14.8%	16.0%
		3.0	0.0%	4.7%	4.1%	6.4%	6.6%	8.4%	11.4%	13.1%	11.7%	12.9%
Intact PTH> 500 pg/mL	Intervention	2.5	0.0%	0.0%	0.0%	0.0%	0.0%	0.9%	1.0%	1.1%	0.0%	1.2%
		3.0	0.0%	0.0%	0.0%	0.0%	0.4%	0.0%	0.4%	0.0%	0.5%	0.9%
	Control	2.5	0.0%	0.0%	0.0%	0.0%	0.0%	1.0%	0.0%	0.0%	1.1%	0.0%
		3.0	0.0%	0.0%	0.0%	0.0%	0.0%	0.8%	0.0%	0.4%	0.0%	0.0%

The prevalence of each laboratory abnormality was calculated by the number of patients with the abnormality divided by the number of patients with the measurement at each study month. Abbreviations: D-Ca, dialysate calcium; M, month; PTH, parathyroid hormone.

Fig 1

Serial changes in the median (25th, 75th percentile) of serum levels of corrected Ca, phosphate, and intact PTH in D-Ca 2.5 and 3.0 mEq/L.

In the stratified analyses by D-Ca, treatment with alfacalcidol was not significantly associated with the composite cardiovascular events, all-cause mortality, or the combined outcome in either D-Ca concentration (Table 2). The associations of treatment with alfacalcidol with these outcomes were not significantly modified by D-Ca (Fig 2).

Table 2

Estimated association of alfacalcidol use with the two outcomes stratified by dialysate calcium concentration.

Exposure	Outcome	Strata	Hazard Ratio (95% CI)	P-value
Alfacalcidol (use vs. nonuse)	Composite CVD events	D-Ca 2.5 mEq/L	1.119 (0.626–1.997)	0.705
		D-Ca 3.0 mEq/L	1.066 (0.703–1.619)	0.762
	ACM	D-Ca 2.5 mEq/L	1.047 (0.523–2.099)	0.896
		D-Ca 3.0 mEq/L	0.710 (0.440–1.147)	0.161
	Composite CVD events or ACM	D-Ca 2.5 mEq/L	1.087 (0.651–1.815)	0.749
		D-Ca 3.0 mEq/L	0.910 (0.638–1.298)	0.602

Abbreviations: CVD, cardiovascular disease; ACM, all-cause mortality; D-Ca, dialysate calcium concentration; CI, confidence interval.

Fig 2

Estimated hazards of composite cardiovascular events (primary outcome), all-cause mortality (secondary outcome), and the combined outcome (primary plus secondary) by the use of alfacalcidol and the baseline dialysate calcium concentration.

Estimated hazard was plotted against the dialysate calcium concentration stratified by the use of alfacalcidol.

Also, in the stratified analyses by treatment with alfacalcidol, the D-Ca was not significantly associated with the composite cardiovascular events, all-cause mortality, or the combined outcome regardless of treatment with alfacalcidol (Table 3). The associations of D-Ca with these clinical outcomes were not significantly modified by the use of alfacalcidol (Fig 2).

Table 3

Estimated association of dialysate calcium concentration with the two outcomes stratified by use of alfacalcidol.

Exposure	Outcome	Strata	Hazard Ratio (95% CI)	P-value
D-Ca (3.0 vs 2.5 mEq/L)	Composite CVD events	Alfacalcidol use (–)	1.093 (0.665–1.795)	0.727
		Alfacalcidol use (+)	1.042 (0.636–1.705)	0.871
	ACM	Alfacalcidol use (–)	1.315 (0.735–2.353)	0.356
		Alfacalcidol use (+)	0.891 (0.507–1.566)	0.689
	Composite CVD events or ACM	Alfacalcidol use (–)	1.179 (0.763–1.821)	0.458
		Alfacalcidol use (+)	0.986 (0.649–1.499)	0.949

Abbreviations: CVD, cardiovascular disease; ACM, all-cause mortality; D-Ca, dialysate calcium concentration; CI, confidence interval.

Discussion

The present study showed that the D-Ca in the range of 2.5 to 3.0 mEq/L did not have statistically significant impact on the primary and secondary outcomes of the J-DAVID participants in either the users or non-users of alfacalcidol. Other than D-Ca, many factors such as medication, diet, bone status, and ultrafiltration volume are involved in the calcium balance of HD patients. Optimal D-Ca has been discussed before [11, 12] and after [13, 14] the introduction of calcimimetics (calcium-sensing receptor agonists). In the recent KDIGO CPG, D-Ca with the range of 2.5 to 3.0 mEq/L was suggested [1, 15]. Within this range of D-Ca, the effect of alfacalcidol on clinical outcomes was not significantly modified.

One may speculate that treatment with VDRA is harmful in patients without secondary hyperparathyroidism who are dialyzed against higher concentrations of D-Ca because calcium load is higher. However, we noticed a numerically higher hazard for the composite of cardiovascular events associated with a higher D-Ca in patients who were not treated with alfacalcidol, not in patients who were treated with alfacalcidol, although the result was not statistically significant. The same was true in the association of D-Ca with the hazard for all-cause mortality. Although the exact mechanisms for these observations are unknown, the calcium load from dialysate may not have a detectable impact on clinical outcomes in the range of 2.5 to 3.0 mEq/L of D-Ca. According to the JSDT data in 2009, D-Ca was 2.5 to 3.0 mEq/L in more than 96% of the total number of reported dialysis patients (N = 209,322) in Japan. Otherwise, the result might have been affected the fact that intravenous VDRAs other than oral alfacalcidol were allowed to be used if needed to follow the JSDT guideline, and that more patients in the control group received intravenous VDRAs than the intervention group of the original J-DAVID trial.

This post hoc analysis was not able to show statistically significant association of D-Ca with composite cardiovascular events or all-cause mortality. The lack of significant associations may be attributable to the relatively low statistical power of the original J-DAVID trial. To address this issue, we took another outcome including both the primary composite cardiovascular outcome and the secondary all-cause mortality in this pos hoc analysis. However, the association of D-Ca and the combined outcome was again not significant. Similar to our results, another observational study derived from the Japanese Dialysis Outcomes and Practice Patterns Study reported that the difference in D-Ca was not related to the survival based on the results of 9,201 patients with a median follow-up of 2.03 years [16]. Thus, the low statistical power does not fully explain the neutral results on the D-Ca in this range and clinical outcomes in this study.

Kim HW et al reported that high D-Ca (3.5 mEq/L) was associated with a higher mortality rate and hospitalization with CVD or infection among incident HD patients [17]. “There are potential safety concerns associated with the default use of dialysate calcium concentrations (2.50 mEq/L)” [18]. High D-Ca (3.5 mEq/L) has a higher risk of coronary artery calcification than that lower D-Ca (2.5 mEq/L) [19]. Tagawa et al reported that High (3.0 mEq/L and over) was associated with incident MI among DM dialysis patients with low bone turnover [20].

Control of serum levels of Ca, phosphate, and PTH is mandatory for the prevention and management of CKD-MBD. Hyperphosphatemia is independently associated with an elevated risk of sudden death in patients on hemodialysis [21]. Yamada S et al reported that high serum calcium (10.0–16.5 mg/dL) is a risk factor of infection-related and all-cause death in hemodialysis patients [22]. Concerning the treatment of hyperphosphatemia either non-Ca-based or Ca-based phosphate binder, Ogata H et al compare the effects between non-calcium-based phosphate binders and calcium-based binders for reducing cardiovascular events. There was no difference in all-cause mortality among Japanese HD patients. However, the results may be confounded by the relatively low incidence of cardiovascular events among Japanese patients [23].

Sakoh T et al conducted a short-term (6 months) intervention study to see the effect of changing D-Ca, from 3.0 to 2.75 mEq/L (N = 12) and 2.5 to 2.75 mEq/L (N = 12) [2]. Although the intradialytic Ca loading was different, there were no significant differences in serum Ca, phosphate, PTH, and fibroblast growth factor 23. Yamada S et al observed the effects of lowering D-Ca from 3.0 to 2.75 mEq/L [24]. One year after the conversion, the mean serum Ca level decreased, while serum phosphate, alkaline phosphatase, and PTH increased. These authors concluded that D-Ca should be individualized based on clinical factors. However, a single patient dialysis fluid delivery system (SPDDS) is very uncommon in Japan as the central dialysis fluid delivery system (CDDS) is safe and evolved for more than 50 years [3].

Calcimimetic agents (calcium-sensing receptor agonists) have been available since January 2008 in Japan. During the recruiting period, July 1, 2008, to January 26, 2011, cinacalcet (the first calcimimetic agent in the Japanese market) was used at the start of the J-DAVID trial in 5.9% of the total participants [9]. Most of the dialysis units followed the JSDT guideline published in 2008 [25] and updated in 2013 accordingly [26]. Both cinacalcet and evocalcet are effective for controlling secondary hyperparathyroidism [27] irrespective of D-Ca [28, 29]. By comparing the effects on serum calcification propensity (T50), a surrogate marker of calcification, Shoji T et al reported that a calcimimetic agent etelcalcetide was more effective than a vitamin D receptor activator maxacalcitol [30].

Regular monitoring of serum levels of calcium, phosphate, and PTH is mandatory for the control of CKD-MBD and improving survival. Large differences in these parameters among countries and races, yet the potential confounders of these observations remained to be studied. [31]. However, precise control of serum phosphate and calcium may achieve adequate levels of PTH [32-35]. The target of PTH is low as 60 to 180 pg/ml and the survival is excellent in Japan [36].

The strengths of the present study are the post hoc analysis of the J-DAVID trial; therefore, the observation period is relatively long, with a median of 4 years, and the follow-up rate was quite high. Serial laboratory data on mineral metabolism such as corrected Ca, phosphate, and intact PTH were available. The rates of exclusion and loss to follow-up were small as 1.2% and 2.0%, respectively. However, there are several limitations in the present study. Firstly, the study subjects were those with no evidence of severe secondary hyperparathyroidism whose baseline intact PTH levels were 180 pg/mL or lower. Therefore, we could not examine the possible effects of alfacalcidol and different D-Ca levels and the interactions on the clinical outcomes. Second, the vintage of HD was relatively short as the median of 5.6 years. Third, dialysate composition other than calcium was not considered. Finally, the statistical power to detect the differences if any was small as stated in the original paper [4]. This is partly due to the number of events being small.

In conclusion, we observed no significant effects of D-Ca on the CKD MBD parameters and the clinical outcomes. Results support the notion that the current strategy of using D-Ca between 2.5 to 3.0 mEq/L is safe. Further research is necessary for those with secondary hyperparathyroidism.

(CSV)

Click here for additional data file.

31 Mar 2022

18 May 2022

Response to comments by the Editor and the two Reviewers

1. Our response to comments by the editor

We appreciate the time and effort of the editor in evaluating our manuscript.

Editor comment #1.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Our reply

We did it as required. We have combined the files for each part of the figure, and now, the file names for figures are Fig1.pptx, Fig2.pptx. Because it is difficult for us to convert file format, can you kindly convert these pptx files into the format appropriate for PLOS One, please?

Editor comment #2.

Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Our reply

All the participants of the J-DAVID trial gave written informed consent. This has been indicated in the revised manuscript. In this revision, we would like to rewrite this part in order to mention the approvement of the original J-DAVID trial by ethics committee. Please see Change #5 in the list of changes below.

Editor comment #3.

We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Our reply

The information in ‘Financial Disclosure’ section is correct, and it has not been changed during this revision. This study and the original J-DAVID trial were supported by the grant to TS from The Kidney Foundation, Japan as indicated in the original version of this manuscript. The grant number was not indicated, because this grant had no specific grant number. Therefore, in response to the above comment, we have added that this grant had no specific number (Change #11). Some coauthors disclosed research grant, but all these grants were not related to the J-DAVID trial or the post hoc analysis.

Editor comment #4.

Thank you for stating the following in the Competing Interests section:

"Kunitoshi Iseki received personal fees from Kyowa Kirin, Bayer Yakuhin, Torii Pharmaceutical, Mochida Pharmaceutical, and Kissei Pharmaceutical; and received consultation fee from the MediGate Digital Health K.K., Daijiro Kabata received personal fees from Chugai Pharmaceutical; research grant and consultation fee for statistical analysis from Kyowa Kirin; and personal fees and a research grant from Bayer Yakuhin outside the submitted work. Ayumi Shintani received personal fees from Chugai Pharmaceutical, Bayer Yakuhin, and Ono Pharmaceutical; and personal fees and research grants from Kyowa Kirin, Takeda Pharmaceutical, and Daiichi Sankyo outside the submitted work. Shinya Nakatani received personal fees from Chugai Pharmaceutical, Kyowa Kirin, Bayer Yakuhin, Ono Pharmaceutical, Kissei Pharmaceutical, and Torii Pharmaceutical. Tomoaki Morioka received personal fees from Ono Pharmaceutical. Katsuhito Mori received personal fees from Ono Pharmaceutical; he served as the principal investigator of a clinical trial for Kyowa Kirin; and personal fees and a research grant from Mitsubishi Tanabe outside the submitted work. Masaaki Inaba received personal fees from Chugai Pharmaceutical, Kyowa Kirin, Bayer Yakuhin, Ono Pharmaceutical, Kissei Pharmaceutical, and Torii Pharmaceutical outside the submitted work. Masanori Emoto received personal fees from Kissei Pharmaceutical, AstraZeneca, and Torii Pharmaceutical; and personal fees and research grants from Chugai Pharmaceutical, Kyowa Kirin, Ono Pharmaceutical, Nippon Boehringer Ingelheim, and Mitsubishi Tanabe outside the submitted work. Tetsuo Shoji received personal fees from Chugai Pharmaceutical, Kyowa Kirin, and Kissei Pharmaceutical; and personal fees and research grants from Bayer Yakuhin, and Ono Pharmaceutical outside the submitted work."

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Our reply

1We have added the statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials” in the Competing Interests section （Change #12）. Data sharing statement is also added （Change #13）. The competing interests information written in the manuscript is correct and no update is needed. As suggested, we have included the Competing interests information in the cover letter. Can you handle it appropriately in the editorial office, please?

Editor comment #5.

Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files"

Our reply

We have included our tables as part of our main manuscript （Changes #14, #15）and removed the individual files which were originally submitted in PPT files. Also, supplementary tables have been handled as suggested.

Editor comment #6.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Our reply

We have reviewed the reference list and confirmed that it is correct. No change has been made to it during revision.

2. Our response to comments by Reviewer #1

We appreciate the time and effort of the reviewer in evaluating our manuscript.

Reviewer #1, Comment #1

Another nice publication out of J-DAVID trial.

As with the previous J-DAVID publication in JAMA (JAMA. 2018;320(22):2325–2334), I have difficulty understanding the definition of "HD patients without secondary hyperparathyroidism" that they chose "PTH of less than or equal to 180 pg/ml."

Although we typically do not use VDRA in those patients, the PTH of 180 pg/ml is close to 3times upper limit of normal and in most labs PTH of > 80 pg/ml is considered secondary hyperparathyroidism. A better term might be mild secondary hyperparathyroidism that guidelines does not require VDRA treatment.

In the previous JAM article they mention: "...Compared with the control group, the intervention group had lower proportions of calcium carbonate users and cinacalcet users, and higher proportions of sevelamer users and lanthanum carbonate users. The proportion of intravenous VDRA users increased over time in both groups, although the proportion was higher in the control group." That raises many question about the study for me:

Our reply .

This comment includes two different points, namely, 1-1. The term which defined the participants who had intact PTH of 180 pg/mL or lower, and 1-2. The potential problems which were introduced by the use of IV-VDRA in the two groups in the original J-DAVID trial.

1-1. We agree that the term “secondary hyperparathyroidism” used in the original J-DAVID trial may be somewhat confusing. According to the 2006 JSDT guideline, the recommended range of intact PTH was 60-180, which was higher than the reference range of intact PTH which is set for “normal” individuals not on dialysis. However, although the target range of intact PTH was set by the JSDT guideline and other clinical practice guidelines, there is no clear DEFINITION of secondary hyperparathyroidism (SHPT) for patients on dialysis. Therefore, we used the term SHPT in the original JAMA paper to mean “elevated intact PTH which needs treatment” namely PTH levels over the target range. In the clinical practice of hemodialysis patients, PTH level in this range was considered appropriate and NOT regarded as “hyperparathyroidism”. The use of this term was not criticized by any reviewers or editors of JAMA. Therefore, although we agree that it may be somewhat confusing, we do not believe that the term is not wrong.

In response to this comment, we have avoided the expression of “patients without secondary hyperparathyroidism” in the Abstract. Please see Change #1 in the list of changes below.

1-2. We appreciate this question. We would like to point out the difficulty in clinical trials in CKD-MBD. There are many players in CKD-MBD such as calcium, phosphate, PTH, vitamin D, FGF23, and others. All of these are closely linked in a complex manner. Therefore, if one of these factors is intentionally changed in a clinical trial, other components will change accordingly. Therefore, it was ethically needed to permit the use of some drugs other than the test drug alfacalcidol. In cases in whom SHPT developed even treated with alfacalcidol, it may be needed to change alfacalcidol to more potent intravenous VDRA to follow the clinical practice guideline. This was also the case with the patients in the control group. If no VDRA was given, secondary hyperparathyroidism would have been more advanced as its natural course. Thus, some rescues other than the test drug were ethically needed in the control group. This difficult situation of CKD-MBD trials is quite different from that of trials with a statin.

When the J-DAVID trial was open, cinacalcet was not available in Japan. It was used during the recruitment period used in Japan. Therefore, investigators in each site used IV VDRA to control SHPT if needed.

Therefore, the J-DAVID trial could not evaluate the pure pharmacological effect of alfacalcidol, but the results of the J-DAVID indicate that the “VDRA first” strategy did not result in better clinical outcomes in hemodialysis patients with intact PTH of 180 pg/mL or lower as compared to the “VDRA last” strategy.

Since we supposed the use of IV-VDRA in the J-DAVID trial, we pre-planned analyses which considered the use of IV-VDRA, in addition to the primary ITT analysis using the full analysis set (FAS). Namely, analyses using per-protocol set (PPS) and modified per-protocol set (m-PPS). In PPS, patients who switched from oral alfacalcidol to IV-VDRA in the intervention group were censored, and patients who used IV-VDRA in the control group were censored, at the time of IV-VDRA use. In m-PPS, patients who used IV-VDRA in the control group were similarly censored at the time of IV-VDRA use, whereas patients who switched from oral alfacalcidol to IV-VDRA in the intervention group were not censored because they were kept treated with any VDRA. Very interestingly, these analyses with PPS and m-PPS showed higher hazard ratios than the primary analysis, and the P-values were lower. The analysis with m-PPS gave HR of 1.36 (0.99-1.87), P=0.06, when adjusted for age, sex, diabetic kidney disease, dialysis vintage, prior CVD, and facilities (Please see Table 3 of the JAMA paper).

Thus, the use of IV-VDRA in the control group introduced contamination of treatment in the trial and reduced the statistical power of this trial. When the effect of contamination was carefully removed, the use of alfacalcidol and other IV-VDRA appeared to increase the risk of CVD in this population, although it was not statistically significant.

In response to this comment, the possible influence of IV-VDRA used in the control group has been discussed in the revised manuscript (Change #10).

Reviewer #1, Comment #2.

Why did they used IV VDRA or cinacalcet (I assume much rise in PTH), and what happened to comparisons in those who did get IV VDRA and those who did not?

Our reply

We appreciate this comment. According to the protocol of the original J-DAVID trial, IV-VDRAs other than the test drug (oral alfacalcidol) were allowed to be used if needed to follow the JSDT guideline. Therefore, although we recorded the exact reason for the use of IV-VDRA, we also assume that these patients experienced an increase in intact PTH which needed intensified medical treatment. Although we did not compare the clinical outcomes between those who took IV-VDRA and those who did not, the possible effects of the use of IV-VDRA in the control group and in the intervention group were addressed in additional analyses using PPS and m-PPS as mentioned above. In response to this comment, we have mentioned the possible reason for the use of IV-VDRA in the revised manuscript (Change #10).

Reviewer #1, Comment #3.

The main reason most guidelines, including KDIGO avoid lowering PTH more and use of VDRA is not required in PTH of <180 pg/ml, is to avoid the low bone turnover and dynamic bone which is a disaster to treat. Higher calcium and even lower PTH were seen initially in both high D-Ca groups and VDRA users in J-DAVID trial. Did they look into the adverse effects of potential low bone turnover in these groups?

Our reply

We appreciate this comment. We did not examine whether bone turnover affected the clinical outcome in the original J-DAVID trial. However, bone turnover may modify the effect of alfacalcidol on the clinical outcomes as pointed out by the reviewer. Therefore, we have performed another post hoc analysis using ALP activity as an index of bone turnover, and we found that the effect modification was not statistically significant. The results are currently in the review process in another journal.

In response to this comment, we did not make changes in this manuscript.

We appreciate again the careful evaluation and constructive comments by the reviewer.

Our response to comments by Reviewer #2:

We appreciate the time and effort of the reviewer in evaluating our manuscript.

Reviewer #2, Comment #1

The study by Iseki et al is a very nice posthoc analysis of the J-DAVID trial is very nicely written. The authors compared 2.5 and 3 mEq/L of dialysate calcium and hwo they responded to treatment verse control with VDRA. I have a couple concerns about the study that I would like the authors to address:

1) The authors did not discuss the power of seeing a difference with treatment. The original study reported that you needed a sample size of 1600 to see a difference between groups with an expected primary outcome of 32% in the control group. However the authors only had 333/331 individuals in the 3.0 mEq/L Ca. However, for the primary outcome of mortality the 3.0 group had a hazard ratio of 0.710 with a p value of 0.16. The authors should discuss the fact that intervention may have an affect on mortality in those dialyzed against a higher Ca, but they may have been underpowered.

We appreciate this comment. We agree with the reviewer. The lack of sufficient statistical power was already mentioned as one of study limitations in the original manuscript. We have discussed this issue in a new paragraph in the revised manuscript. Please see Change #10. in the list of changes below.

Reviewer #2, Comment #2

Table 1a it is confusing. Instead of putting in Number of reported values under abnormalities. Maybe change it to all reported calcium, PTH and phosphate?

We agree that Table 1a was confusing. The same is true for Table 1b, Table 1c, and Table 1d. There were so many numbers in the table which may not be necessary for the readers. In response to the advice to remove “Number of reported values” from the column of “abnormalities”, we have revised these tables to be more simple and more concise (Change #14). Similarly, Table 2a and Table 2b have been edited into Table 2 and Table 3 (Change #15). We believe that these tables have become much more readable after revision.

Reviewer #2, Minor concern #1

Line 32 please change oral alfacalcidol to vitamin D receptor agonist (oral alfacidol)

We appreciate this comment. Although we used “vitamin D receptor agonist”, “vitamin D receptor activator” is more common for the full spelling of VDRA. Therefore, we have changed it to a vitamin D receptor activator (Change #1).

Reviewer #2, Minor concern #2

Line 56 please change vitamin receptor agonist to vitamin D receptor agonist

We have corrected this part (Change #2).

Reviewer #2, Minor concern #3

Last line page 3, lines 77-78. Needs to be reworded.

We corrected it as suggested. (Change #3)

Reviewer #2, Minor concern #4

The result section should all be changed to present tense rather than past tense.

We do not agree with this advice. It is standard to describe the results in the past tense rather than present tense.

Reviewer #2, Minor concern #5

Line 112 the he should be changed to the

We appreciate your very careful evaluation. Our careless mistake "he" has been changed to "the". But this part of the text has been further revised to be more readable (Change #7).

We appreciate again the careful evaluation and constructive comments by the reviewer.

List of changes

Change #1

Original, Page 2, Abstract, Line 31-32:

The trial was an open-label randomized clinical trial including 976 HD patients without secondary hyperparathyroidism which compared the users of oral alfacalcidol and non-users over a median of 4 years.

Revised, Page, Line :

The trial was an open-label randomized clinical trial including 976 HD patients with intact PTH of 180 pg/mL or lower which compared the users of vitamin D receptor activator (oral alfacalcidol) and non-users over a median of 4 years.

Change #2

Original, Page 3, Line 56-57:

Many observational studies showed that the use of vitamin receptor agonist (VDRA) was associated with a lower risk of all-cause mortality (4, 5),

Revised, Page 3, Line 56-57:

Many observational studies showed that the use of vitamin D receptor activator (VDRA) was associated with a lower risk of all-cause mortality (4, 5),

Change #3

Original, Page 3, Line 77-80:

The number and percentage of patients were too small as 5% with D-Ca 2.75 mEq/L and 2.9 mEq/L. Therefore, we obtained additional data such as corrected calcium, intact PTH, and serum phosphate according to the D-Ca of 2.5 and 3.0 mEq/L.

Revised, Page 3-4, Line 78-80:

Majority of patients (95%) used D-Ca of 2.5 mEq/L or 3.0 mEq/L, and only the small proportion of patients used D-Ca of 2.75 mEq/L or 2.9 mEq/L. Therefore, comparison was done between the two groups of D-Ca of 2.5 and 3.0 mEq/L.

Change #4

A new paragraph has been included in the Methods section, 2nd paragraph to explain the reason why the new combined outcome was considered.

Revised, Page 4, Line 81-84:

In the original J-DAVID trial, the primary outcome was the composite of fatal and non-fatal cardiovascular events (N = 188), and the secondary outcome was all-cause mortality (N = 169). To increases the power of statistical analysis, this post hoc analysis additionally considered the combined outcome including both the primary and secondary outcomes (N = 277).

Change #5

Original, Page 4, Line 94-95:

The institutional review board of the Osaka City University approved the research project, Number 4420 on September 26, 2019.

Revised, Page 4-5, Line 98-106:

The J-DAVID trial was conducted in accordance with the principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Studies by the Ministry of Health, Labor and Welfare, Japan (the original 2003 version, which was modified in 2004 and 2006). The protocol and revisions of this trial were approved by the ethics committee at the Osaka City University Graduate School of Medicine in Japan (approval numbers 1227, 1297, 1385, and 1525) and by the relevant ethics committees or institutional review boards at the study sites. All participants gave written informed consent before the study. The protocol of post hoc analyses using the original J-DAVID trail was reviewed and approved by the ethics committee of the Osaka City University Graduate School of Medicine (Number 4420 on September 26, 2019).

Change #6

Original, Page 4, Line 100-103:

In Table 1a, 1b, 1c, and 1d, the trends of serum levels of corrected Ca, intact PTH, phosphate, and in both D-Ca 2.5 and 3.0 mEq/L were shown. Figures 1a, 1b, and 1c showed the mean (SD) of corrected Ca, phosphate, and intact PTH in both D-Ca 2.5 and 3.0 mEq/L. Although all three variables were within the target ranges, …

Revised, Page 5, Line 111-113:

In Table 1 and Figure 1, the trends of serum levels of corrected Ca, intact PTH, phosphate, and in both D-Ca 2.5 and 3.0 mEq/L were shown. Although all three variables were within the target ranges in most patients, …

Change #7

Original, Page 5, Line 107-114:

In the stratified analyses by D-Ca, the allocation group was not significantly associated with the composite of fatal and non-fatal cardiovascular events in either D-Ca concentration. Also, the D-Ca was not significantly associated with the composite of fatal and non-fatal cardiovascular events in either allocation group. (Table 2a). There were no significant differences between the groups by using interaction (Figure 2).

There were no significant differences between the groups by using interaction he risks hazards of all-cause mortality by D-Ca (Table 2b). There were no significant differences between the groups by using interaction (Figure 3).

Revised, Page 5, Line 116-123:

In the stratified analyses by D-Ca, treatment with alfacalcidol was not significantly associated with the composite cardiovascular events, all-cause mortality, or the combined outcome in either D-Ca concentration (Table 2). The associations of treatment with alfacalcidol with these outcomes were not significantly modified by D-Ca (Figure 2).

Also, in the stratified analyses by treatment with alfacalcidol, the D-Ca was not significantly associated with the composite cardiovascular events, all-cause mortality, or the combined outcome regardless of treatment with alfacalcidol (Table 3). The associations of D-Ca with these clinical outcomes were not significantly modified by the use of alfacalcidol (Figure 2).

Change #8

Original, Page 5, Line 118-120:

The present study showed that the D-Ca in the range of 2.5 to 3.0 mEq/L did not impact the primary and secondary outcomes of the J-DAVID participants both the users and non-users of VDRA.

Revised, Page 5, Line 127-129:

The present study showed that the D-Ca in the range of 2.5 to 3.0 mEq/L did not have statistically significant impact on the primary and secondary outcomes of the J-DAVID participants in either the users or non-users of alfacalcidol.

Change #9

Treatment with alfacalcidol was not based on the allocation group, but it was handled as a time-varying variable in this post hoc analysis, our careless mistake has been corrected.

Original, Page 5, Line 128-130:

However, we noticed a numerically higher hazard for the composite of cardiovascular events associated with a higher D-Ca in patients who were allocated to the control group (without VDRA), not in patients who were allocated to the intervention group (using alfacalcidol), …

Revised, Page 6, Line 137-139:

However, we noticed a numerically higher hazard for the composite of cardiovascular events associated with a higher D-Ca in patients who were not treated with alfacalcidol, not in patients who were treated with alfacalcidol, …

Change #10

We have discussed on the possible influences of intravenous VDRAs and statistical power on the results in the revised manuscript.

Original, Page 6, Line 132-137:

Although the exact mechanisms for these observations are unknown, the calcium load from dialysate may not have a detectable impact on clinical outcomes in the range of 2.5 to 3.0 mEq/L of D-Ca.

According to the JSDT data in 2009, D-Ca was 2.5 to 3.0 mEq/L in more than 96% of the total number of reported dialysis patients (N=209,322) in Japan. Another observational study reported that the difference in D-Ca was not related to the survival similar finding to this study. (16)

Revised, page 6, Line 140-157:

Although the exact mechanisms for these observations are unknown, the calcium load from dialysate may not have a detectable impact on clinical outcomes in the range of 2.5 to 3.0 mEq/L of D-Ca. According to the JSDT data in 2009, D-Ca was 2.5 to 3.0 mEq/L in more than 96% of the total number of reported dialysis patients (N=209,322) in Japan. Otherwise, the result might have been affected the fact that intravenous VDRAs other than oral alfacalcidol were allowed to be used if needed to follow the JSDT guideline, and that more patients in the control group received intravenous VDRAs than the intervention group of the original J-DAVID trial.

This post hoc analysis was not able to show statistically significant association of D-Ca with composite cardiovascular events or all-cause mortality. The lack of significant associations may be attributable to the relatively low statistical power of the original J-DAVID trial. To address this issue, we took another outcome including both the primary composite cardiovascular outcome and the secondary all-cause mortality in this pos hoc analysis. However, the association of D-Ca and the combined outcome was again not significant. Similar to our results, another observational study derived from the Japanese Dialysis Outcomes and Practice Patterns Study reported that the difference in D-Ca was not related to the survival based on the results of 9,201 patients with a median follow-up of 2.03 years (16). Thus, the low statistical power does not fully explain the neutral results on the D-Ca in this range and clinical outcomes in this study.

Change #11

Original, Page 8, Line 197:

Funding: This study was supported by the grant to TS from The Kidney Foundation, Japan.

Revised, Page 9, Line 217-218:

Funding: This study was supported by a grant to TS from The Kidney Foundation, Japan (no specific grant number was given).

Change #12

In the part of Competing interests, we have added the following statement.

Revised, Page 10, Line 245:

This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Change #13

Data sharing statement has been included in the revised manuscript.

Revised, Page 10, Line 246-247:

Data sharing: The data that support the findings of this study are available from the corresponding author of the original J-DAVID trial (TS) upon reasonable request.

Change #14

Tables 1a, 1b, and 1c in the original manuscript has been combined into Table 1 in the revised version. The important contents in the original big tables have been edited to be more concise. The revised Table 1 has been included in the main body of the manuscript.

Change #15

Tables 2a and 2b in the original manuscript has been edited into Table 2 and Table 3 in the revised version. In the original version, the tables were created according to the outcome. In this revision, these tables were re-organized according to the exposure. The revised tables have been included in the main body of the manuscript.

Change #16

Our careless mistake (not mean but median) in the figure legend has been corrected.

Original, Page 13, Line 331-332:

Figure 1. Serial changes in the mean (25%, 75%) of serum levels of corrected Ca (1a), phosphate (1b), and intact PTH (1c) in D-Ca 2.5 and 3.0 mEq/L.

Revised, Page 18, Line 367-368:

Figure 1. Serial changes in the median (25th, 75th percentile) of serum levels of corrected Ca, phosphate, and intact PTH in D-Ca 2.5 and 3.0 mEq/L.

Change #17

Because Figures 2 and 3 in the original version have been combined into new Figure 2 in this revision, Figure numbers and legends have been updated accordingly.

Original, Page 13-14, Line 333-338:

Figure 2. Estimated hazards of all-cause mortality and non-fatal cardiovascular disease events by the use of alfacalcidol and the baseline dialysate Ca. There were no significant differences in the effects depending on the baseline dialysate Ca value. Treat: VDRA (+), control: VDRA (-)

Figure 3. Estimated hazards of all-cause mortality by the use of alfacalcidol and the baseline dialysate Ca. There were no significant differences in the effects depending on the baseline dialysate Ca value. Treat: VDRA (+), control: VDRA (-)

Revised, Page 18, Line 369-372:

Figure 2. Estimated hazards of composite cardiovascular events (primary outcome), all-cause mortality (secondary outcome), and the combined outcome (primary plus secondary) by the use of alfacalcidol and the baseline dialysate calcium concentration. Estimated hazard was plotted against the dialysate calcium concentration stratified by the use of alfacalcidol.

Submitted filename: 2 Response to Editor & Reviewers_0425.docx

Click here for additional data file.

4 Aug 2022

Dialysate Calcium, Alfacalcidol, and clinical Outcomes: A Post-Hoc Analysis of the J-DAVID Trial

PONE-D-22-04275R1

Dear Dr. Kunitoshi Iseki ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Larry Allan Weinrauch, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

26 Aug 2022

PONE-D-22-04275R1

Dialysate Calcium, Alfacalcidol, and clinical Outcomes: A Post-Hoc Analysis of the J-DAVID Trial

Dear Dr. Iseki:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Larry Allan Weinrauch

Academic Editor

PLOS ONE