Vitamin D3 and Body Composition Association with Graft Function in Long-Term Kidney Transplant Recipients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors: The area of calcium. phosphorus, PTH. and Vitamin D are an understudied area of transplantation. Though essentially a negative study, the data provided in the manuscript are quite informative.  The only consideration for future studies (as mentioned in the discussion) might be to include bone density studies. 

Author Response

Comment 1 [Dear Authors: The area of calcium. phosphorus, PTH. and Vitamin D are an understudied area of transplantation. Though essentially a negative study, the data provided in the manuscript are quite informative.  The only consideration for future studies (as mentioned in the discussion) might be to include bone density studies. ]

Response 1 [

Dear Reviewer, we would like to thank the Reviewer for this insightful comment. We agree that bone mineral density (BMD) is a crucial parameter in the context of mineral and bone disorder after kidney transplantation. Nevertheless, the primary objective of this study was to evaluate the associations between vitamin D3 levels, body composition (measured by BIA), and graft function in the long-term follow-up. Including BMD data would indeed expand the scope, but as the Reviewer correctly noted, it could also divert attention from the main focus of our current analysis – which is the interplay between nutritional status (body composition) and graft function. We have addressed this point in the Limitations section of the Discussion, emphasizing that future prospective trials should integrate densitometry to provide a more holistic view of bone health.]

Reviewer 2 Report

Comments and Suggestions for Authors

Vitamin D metabolism can be assessed by two parameters: calcidiol (vitamin D) and calcitriol (hormone D) concentrations. For kidney transplant patients, the latter parameter is more important, as it influences calcium-phosphorus metabolism and has pleiotropic effects. The authors divided patients into three groups based on serum calcidiol concentrations, which is fundamentally incorrect. The transformation of calcidiol into calcitriol depends on the activity of 1-alpha-hydroxylase, i.e., on the volume of functioning renal parenchyma. In patients with end-stage renal disease, this volume is reduced, and regardless of the serum calcidiol concentration, adequate transformation of vitamin D into hormone D is impossible.

Kidney transplantation is an important stage in the correction of vitamin D metabolism disorders, as it partially restores the ability to transform vitamin D into hormone D. However, one healthy organ cannot replace two healthy organs, and the transformation is insufficiently active. Therefore, it is impossible to expect a complete restoration of the systemic effects of hormone D. Consequently, cholecalciferol intake cannot replenish the need for hormone D.

The authors used drugs with different mechanisms of action:

- cholecalciferol, whose conversion requires normal 1-alpha-hydroxylase activity;

- alfacalcidol, a synthetic analogue of calcidiol, which also requires normal 1-alpha-hydroxylase activity;

- paricacitol, a selective activator of vitamin D receptors in the parathyroid glands.

Including patients taking different drugs in the same group guarantees bias in statistical analysis. Therefore, it is impossible to interpret the results of methods such as ANOVa, multivariable quantile regression analysis, and cetera unambiguously.

The relationship between graft function and calcitriol levels is unquestionable. However, graft function influences calcitriol levels, not vice versa.

I believe that the article in its current form cannot be published in your respected journal.

Author Response

Comment 1 [

Vitamin D metabolism can be assessed by two parameters: calcidiol (vitamin D) and calcitriol (hormone D) concentrations. For kidney transplant patients, the latter parameter is more important, as it influences calcium-phosphorus metabolism and has pleiotropic effects. The authors divided patients into three groups based on serum calcidiol concentrations, which is fundamentally incorrect. The transformation of calcidiol into calcitriol depends on the activity of 1-alpha-hydroxylase, i.e., on the volume of functioning renal parenchyma. In patients with end-stage renal disease, this volume is reduced, and regardless of the serum calcidiol concentration, adequate transformation of vitamin D into hormone D is impossible.

Kidney transplantation is an important stage in the correction of vitamin D metabolism disorders, as it partially restores the ability to transform vitamin D into hormone D. However, one healthy organ cannot replace two healthy organs, and the transformation is insufficiently active. Therefore, it is impossible to expect a complete restoration of the systemic effects of hormone D. Consequently, cholecalciferol intake cannot replenish the need for hormone D.]

Response 1 [

We thank the Reviewer for this important physiological perspective. We agree that calcitriol is biologically relevant in kidney transplant recipients; however, in clinical practice and according to current guidelines, serum 25(OH)D remains the established marker used to assess vitamin D status and guide nutritional vitamin D supplementation.

Our methodology strictly adheres to current clinical practice guidelines. According to leading medical societies, including the KDIGO (Kidney Disease: Improving Global Outcomes) and ERA (European Renal Association) [1,2], serum 25(OH)D is the most widely accepted and guideline-supported marker of vitamin D status

Current guidelines do not recommend 1,25(OH)2D3 (calcitriol) as a marker for deficiency for several critical reasons:

1. Clinical Non-Specificity: Calcitriol levels can be influenced by various extra-renal conditions (e.g., granulomatous diseases) and are subject to tight homeostatic regulation by PTH and FGF23, making them an unreliable indicator of nutritional vitamin D stores [3].
2. Biological Instability: The short half-life and low circulating concentrations of 1,25(OH)2D3 make its measurement prone to significant analytical error and misinterpretation compared to the stable 25(OH)D3.
3. Guideline Consensus: KDIGO guidelines explicitly state that even in advanced CKD, nutritional vitamin D deficiency should be treated with cholecalciferol (as in the general population) rather than active analogues as the first-line therapy. Therefore, monitoring 25(OH)D3 is the logical and recommended clinical approach.

We thank the Reviewer for this important comment. We agree that conversion of 25(OH)D to 1,25(OH)₂D₃ depends on preserved 1-alpha-hydroxylase activity and, therefore, on functioning renal parenchyma. However, in kidney transplant recipients, successful transplantation is generally associated with at least partial recovery of 1,25(OH)₂D₃ production compared with the pre-transplant uremic state [3–5].

In our cohort, the median eGFR was approximately 48–53 mL/min/1.73 m², indicating that most patients had moderate graft dysfunction, mainly corresponding to CKD stages G3a/G3b, rather than end-stage kidney disease. Therefore, although calcitriol production may remain impaired compared with individuals with two healthy kidneys, complete absence of conversion cannot be assumed in this population.

For this reason, we consider the use of serum 25(OH)D as the grouping variable to be clinically justified and consistent with current guideline-based practice. Nevertheless, we acknowledge that 25(OH)D does not fully reflect active vitamin D metabolism in kidney transplant recipients, and we have clarified this issue in the revised Discussion.

In conclusion, our study was designed to reflect "real-life" clinical conditions and evidence-based standards. Using 25(OH)D3 as the grouping variable ensures that our results are relevant to practicing clinicians and aligned with current nephrological guidelines.

 

•  
• 1. Jørgensen, H.S.; Vervloet, M.; Cavalier, E.; Bacchetta, J.; de Borst, M.H.; Bover, J. The role of nutritional vitamin D in chronic kidney disease–mineral and bone disorder in children and adults with chronic kidney disease, on dialysis, and after kidney transplantation—a European consensus statement. Nephrol. Dial. Transplant. 2025, 40, 797–822.
• 2. KDIGO. 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kid- ney Disease–Mineral and Bone Disorder (CKD-MBD); KDIGO: Brussels, Belgium, 2017. 
• 3. Pike JW, Meyer MB. The unsettled science of nonrenal calcitriol production and its clinical relevance. J Clin Invest. 2020 Sep 1;130(9):4519-4521. doi: 10.1172/JCI141334. PMID: 32716362; PMCID: PMC7456237.
• 4. Evenepoel  P, Naesens  M, Claes  K  et al.  Tertiary ‘hyperphosphatoninism’ accentuates hypophosphatemia and suppresses calcitriol levels in renal transplant recipients. Am J Transplant  2007;7:1193–200. 10.1111/j.1600-6143.2007.01753.x [DOI] [PubMed] [Google Scholar]
• 5. Evenepoel  P, Meijers  BKI, De Jonge  H  et al.  Recovery of hyperphosphatoninism and renal phosphorus wasting one year after successful renal transplantation. Clin J Am Soc Nephrol  2008;3:1829–36. 10.2215/CJN.01310308 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Comment 2 [ The authors used drugs with different mechanisms of action:

- cholecalciferol, whose conversion requires normal 1-alpha-hydroxylase activity;

- alfacalcidol, a synthetic analogue of calcidiol, which also requires normal 1-alpha-hydroxylase activity;

- paricacitol, a selective activator of vitamin D receptors in the parathyroid glands.

Including patients taking different drugs in the same group guarantees bias in statistical analysis. Therefore, it is impossible to interpret the results of methods such as ANOVa, multivariable quantile regression analysis, and cetera unambiguously.]

Response 2 [

We thank the Reviewer for this important and methodologically sound point. We fully acknowledge that the mechanisms of action and effects on calcitriol levels differ substantially between cholecalciferol, alfacalcidol, and paricalcitol.

However, we would like to emphasize that this is a pragmatic, cross-sectional, "real-world" observational study, rather than a strictly controlled interventional trial. In routine nephrological practice, kidney transplant recipients require individualized, heterogeneous therapies to achieve metabolic targets. Our primary objective was to evaluate the actual biological status—the measured serum 25(OH)D3 concentration—and its correlation with graft function at a single time point, regardless of the therapeutic regimen.

Excluding patients based on their specific medication regimen would create an artificial, highly selected cohort that no longer represents the true post-transplant population, thereby undermining the real-world clinical value of our findings. Furthermore, such exclusions would severely reduce our sample size, compromising statistical power and paradoxically introducing a severe selection bias.

To address the Reviewer's valid concern regarding confounding factors without distorting our real-world data, we have taken the following steps in the revised manuscript:

1. We have explicitly added this therapeutic heterogeneity as a Limitation in the Discussion section, noting its potential impact on the absolute interpretation of ANOVA and regression models.
2. We have refined our conclusions to ensure they are presented strictly as real-world associations rather than isolated physiological mechanisms.

We believe this approach preserves the pragmatic value of our study while transparently acknowledging the pharmacological distinctions highlighted by the Reviewer.]

 

Comment 3 [

The relationship between graft function and calcitriol levels is unquestionable. However, graft function influences calcitriol levels, not vice versa.]

Response 3 [

We completely agree with the Reviewer’s statement. In the pathophysiology of chronic kidney disease and post-transplant state, the volume of functioning renal parenchyma (graft function) is the primary determinant of 1-alpha-hydroxylase activity, and thus directly dictates the circulating levels of calcitriol. Lighter graft dysfunction naturally leads to better preservation of calcitriol synthesis. We apologize if our original phrasing implied a definitive, reverse causal relationship (i.e., that vitamin D status actively drives eGFR in this cohort). Because our study has a cross-sectional design, we can only describe associations and correlations, and we cannot establish direct causality or its absolute direction. While active vitamin D signaling pathway is known to exert pleiotropic, anti-fibrotic, and immunomodulatory effects that might theoretically support long-term graft survival, we acknowledge that the primary direction in our findings is that preserved graft function maintains adequate vitamin D metabolism.]

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

This manuscript presents a cross-sectional analysis of the association between vitamin D status, graft function, mineral metabolism, and body composition in a cohort of kidney transplant recipients (KTRs). The relatively large sample size and the inclusion of both 25(OH)D₃ and 1,25(OH)₂D₃ measurements, as well as detailed body composition analysis, represent important strengths. In addition, the use of multivariable quantile regression is appropriate given the distribution of the data. However, despite these strengths, several important issues limit the interpretability and impact of the findings. Substantial revision is required before the manuscript can be considered for publication. The reviewer’s comments are provided below.

Major comments

First, the cross-sectional design of the study inherently precludes any causal inference. While the authors acknowledge this limitation, several statements throughout the Discussion suggest or imply a causal relationship between vitamin D status and graft function. For example, phrases indicating that vitamin D “may support graft function” or “may contribute to progressive graft dysfunction” go beyond what can be concluded from the present data. The interpretation should be revised to consistently reflect associative rather than causal relationships.

Second, the interpretation of the association between 1,25(OH)₂D₃ and eGFR requires further clarification. The observed positive relationship is most likely explained by preserved renal 1α-hydroxylase activity in patients with better graft function, rather than a direct nephroprotective effect of active vitamin D. This issue should be more clearly discussed to avoid potential misinterpretation.

Third, vitamin D supplementation represents a major potential confounder in this study. A substantial proportion of patients received supplementation, including both native and active forms of vitamin D, and the distribution of supplementation differed across vitamin D status groups. This may have significantly influenced circulating vitamin D levels and attenuated or obscured true associations. The authors should adjust for supplementation in multivariable analyses and, if possible, perform subgroup analyses based on supplementation status and type.

Fourth, the study involves a large number of statistical comparisons, yet no correction for multiple testing was applied. While the exploratory nature of the study is acknowledged, the risk of type I error should be more explicitly addressed, and the authors should consider applying appropriate correction methods or clearly justifying their approach.

Fifth, the novelty of the study appears to be somewhat overstated. Although the inclusion of detailed body composition measures is valuable, similar associations between vitamin D status and graft outcomes have been investigated previously. The authors are encouraged to more precisely define the unique contribution of their work.

Minor comments

The Introduction could be shortened to improve focus, particularly in sections describing general vitamin D physiology. Additionally, the Results section could be structured more clearly to improve readability, and the Discussion would benefit from a more in-depth consideration of unexpected findings, such as the paradoxical association between vitamin D deficiency and higher eGFR in certain analyses.

Comments on the Quality of English Language

The manuscript would benefit from careful language editing, as there are several grammatical inconsistencies and awkward phrasings throughout the text.

Author Response

Comment 1 [First, the cross-sectional design of the study inherently precludes any causal inference. While the authors acknowledge this limitation, several statements throughout the Discussion suggest or imply a causal relationship between vitamin D status and graft function. For example, phrases indicating that vitamin D “may support graft function” or “may contribute to progressive graft dysfunction” go beyond what can be concluded from the present data. The interpretation should be revised to consistently reflect associative rather than causal relationships]

Response 1 [We completely agree with the Reviewer’s critical point. As this is a cross-sectional study, we cannot infer causality. We have thoroughly revised the entire Discussion and Conclusion sections to eliminate any causal language. Phrases such as "may support graft function" or "may contribute to progressive graft dysfunction" have been removed or rephrased to strictly reflect associations rather than causal relationships. For instance, these sections now consistently emphasize "metabolic associations" and "co-existing patterns" rather than directional effects.]

 

Comment 2 [

Second, the interpretation of the association between 1,25(OH)₂D₃ and eGFR requires further clarification. The observed positive relationship is most likely explained by preserved renal 1α-hydroxylase activity in patients with better graft function, rather than a direct nephroprotective effect of active vitamin D. This issue should be more clearly discussed to avoid potential misinterpretation.]

Response 2 [We fully concur with the Reviewer’s physiological interpretation. In chronic kidney disease and the post-transplant setting, the volume of functioning renal parenchyma (graft function) is the primary determinant of 1α-hydroxylase activity and thus directly governs circulating calcitriol levels. Accordingly, the observed positive association between 1,25(OH)₂D₃ and eGFR is most likely driven by preserved renal function and intact enzymatic activity in patients with better graft performance, rather than reflecting a primary nephroprotective effect of calcitriol.

We acknowledge that our initial wording may have implied a bidirectional or reverse causal relationship. However, given the cross-sectional design of the study, our results are limited to associations and do not allow inference of causality or its direction. While active vitamin D signaling has pleiotropic anti-fibrotic and immunomodulatory properties that could theoretically support long-term graft outcomes, the primary interpretation supported by our data is that preserved graft function maintains adequate vitamin D metabolism.

To avoid any potential misinterpretation, we have clarified this unidirectional causal pathway in Section 4.1 of the revised Discussion and other relevant parts of the manuscript, emphasizing a graft function–driven rather than vitamin D–driven relationship.]

 

Comment 3 [Third, vitamin D supplementation represents a major potential confounder in this study. A substantial proportion of patients received supplementation, including both native and active forms of vitamin D, and the distribution of supplementation differed across vitamin D status groups. This may have significantly influenced circulating vitamin D levels and attenuated or obscured true associations. The authors should adjust for supplementation in multivariable analyses and, if possible, perform subgroup analyses based on supplementation status and type.]

Response 3 [The Reviewer raises an important and clinically relevant point. In this pragmatic, real-world observational study, we aimed to assess metabolic profiles in a stable, unselected outpatient cohort of kidney transplant recipients under routine clinical care, where concomitant therapies—including native cholecalciferol and active vitamin D analogues—are commonly used. We acknowledge that this heterogeneous treatment background may act as a confounding factor and potentially attenuate observed metabolic associations.

Due to the limited sample size and the heterogeneous distribution of native and active vitamin D preparations, fully powered subgroup analyses according to supplementation type were not feasible. We therefore refrained from performing underpowered subgroup comparisons that could generate unstable estimates. Instead, we expanded the Limitations section and clarified that supplementation may have influenced circulating vitamin D metabolites and attenuated or obscured associations.]

 

Comment 4 [  Fourth, the study involves a large number of statistical comparisons, yet no correction for multiple testing was applied. While the exploratory nature of the study is acknowledged, the risk of type I error should be more explicitly addressed, and the authors should consider applying appropriate correction methods or clearly justifying their approach.]

Response 4 [We appreciate this important comment. We agree that performing multiple comparisons may increase the risk of false-positive findings, especially for results close to the significance threshold. Given the exploratory nature of this study, we did not apply formal correction for multiple testing, as overly conservative correction methods could obscure potentially relevant clinical and metabolic associations. However, we acknowledge that the absence of such correction increases the risk of type I error. We have now explicitly addressed this issue in the Statistical Analysis and Limitations sections, and we emphasize that the findings should be interpreted as descriptive and hypothesis-generating rather than confirmatory.]

 

Comment 5 [Fifth, the novelty of the study appears to be somewhat overstated. Although the inclusion of detailed body composition measures is valuable, similar associations between vitamin D status and graft outcomes have been investigated previously. The authors are encouraged to more precisely define the unique contribution of their work.]

Response 5 [We thank the Reviewer for this guidance and agree that our initial phrasing may have sounded overly broad. 

We agree that the novelty of our study should be stated more precisely and that our initial wording may have overstated this aspect. The unique contribution of our work does not lie in demonstrating a general association between vitamin D status and graft outcomes, as this has been investigated previously.

Rather , it lies in the simultaneous assessment of both 25(OH)D₃ and 1,25(OH)₂D₃ in relation to a highly detailed, multi-compartment body composition assessment (including visceral fat area, soft lean mass, and phase angle via BIA) in a stable cohort with a remarkably long transplant vintage (median 6 years). We have revised the relevant parts of the Introduction and Discussion to define the specific contribution of our study more clearly and to avoid overstating its novelty.]

 

Comment 6 [The Introduction could be shortened to improve focus, particularly in sections describing general vitamin D physiology. Additionally, the Results section could be structured more clearly to improve readability, and the Discussion would benefit from a more in-depth consideration of unexpected findings, such as the paradoxical association between vitamin D deficiency and higher eGFR in certain analyses.]

Response 6 [We thank you for constructive and useful suggestions. We have shortened the Introduction according to your opinion by reducing the generic descriptions of general vitamin D physiology. Moreover, we have improved the readability of the Results section, as seen in the edited manuscript. Regarding the unexpected findings in Section 4.1, we have expanded our discussion on the counterintuitive, paradoxical association between 25(OH)D₃ deficiency and higher eGFR. We have attributed this finding to residual confounding, reverse causality, or specific prescription patterns—whereby clinicians more aggressively prescribe supplements to patients with poorer renal function, leaving healthier individuals unsupplemented and statistically 'deficient'—rather than to a true biological protective effect. ]

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

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