Vitamin D status as a predictor for liver transplant outcomes

It is well known that vitamin D plays a pivotal role in immune system modulation; however, its role in liver transplantation (LT) has not yet been well elucidated. This study aimed to assess the association between vitamin D status and LT outcomes. This retrospective cohort study was conducted on 335 registered cirrhotic patients with end-stage liver disease (ESLD) who underwent LT during 2019–2021 and had measurement of serum vitamin D before LT. The association of vitamin D levels before LT with the odds of acute cellular rejection (ACR) and risk mortality was assessed by applying logistic and cox regression, respectively. The mean MELD-Na and serum level of vitamin D were 20.39 ± 9.36 and 21.52 ± 15.28 ng/ml, respectively. In the final adjusted model, there was a significant association between vitamin D deficiency in the pre-transplant period and odds of ACR (odds ratio [OR] 2.69; 95% confidence interval [CI] 1.50–4.68). Although in the crude model, vitamin D deficiency in the pre-transplant period was significantly associated with an increased risk of mortality after two years of follow-up (Hazard ratio (HR) = 2.64, 95% CI 1.42–4.33), after adjustment for potential confounders, the association of vitamin D status and mortality became non-significant (HR = 1.46, 95% CI 0.71–3.00). The present study provides evidence that pre-transplant serum vitamin D levels may be a predictor for ACR in patients with cirrhosis undergoing LT.


Vitamin D measurement
25-hydroxyvitamin D levels (25(OH)D) were measured along with other laboratory tests before LT.In this study, serum levels of vitamin D under 20 ng/ml were considered deficient, and ≥ 20 ng/ml as sufficient.For vitamin D, we used the most recent lab results available before LT, from which we also obtained other lab results.

MELD and MELD-Na calculation
The Model for End-Stage Liver Disease (MELD) scores were calculated using the following formulas based on the last lab results before LT for bilirubin, creatinine, INR, and Na 24 .Serum sodium value was corrected in the range of 125-137 mEq/l, according to criteria determined by UNOS (United Network for Organ Sharing).

Outcomes identification
In clinical practice, ACR after LT is associated with allograft dysfunction, which is concurrently diagnosed by liver biopsy and histological analysis 25 .Accordingly, when there was suspicion that there may be an ACR reaction, a biopsy was performed in this study.Mortality was also monitored for two years following LT or until death occurred.In addition, CMV infection was detected and diagnosed by CMV antigen-positive peripheral leukocytes.

Statistical analysis
After providing the normality of the distribution of the studied variables by Kolmogorov-Smirnov test, independent sample T-Test used to compare quantitative variables between the two groups, as well as Chi-square or fisher exact statistical test was also used for qualitative variables.The baseline characteristics were reported as mean ± standard deviation (SD) for quantitative variables, and number and percentages for qualitative variables.Regression model was used to examine the correlation between variables.The association of vitamin D levels before LT with the odds of ACR and risk of mortality was assessed by applying logistic and cox regression, respectively.The analyses were adjusted for probable confounders, e.g., age, sex, BMI, waiting time for transplantation, MELD =3.78 × ln serum bilirubin mg/dL + 11.2 × ln [INR] + 9.57 × ln serum creatinine mg/dL + 6.43 time was 4.38 months.In addition, about 19% of the samples were infected with CMV after transplantation, and the prevalence of diabetes and hypertension in the study population was 22.7% and 7.5%, respectively (Table 1).The baseline characteristics of the patients between two groups of subjects with sufficient and deficient levels of vitamin D in the pre-transplant stage are shown in Table 2. Compared to those with sufficient vitamin D levels, those with vitamin D deficiency had higher MELD-Na scores and waiting time for LT but lower albumin levels.In addition, the number of patients with CMV infection after LT was significantly higher in those with vitamin D deficiency.However, no significant differences were found for other characteristics between subjects with sufficient and deficient levels of vitamin D.
Characteristics of patients based on ACR and mortality status after liver transplantation are also indicated in Tables 3 and 4, respectively.
A significant difference was observed between the percentage of patients with hypertension and medical conditions at the time of LT between two groups of patients with and without ACR after LT.Furthermore, the findings of Table 4 showed that age, MELD-Na score, waiting time for LT, FBS, and WBC levels were significantly www.nature.com/scientificreports/higher in patients with post-transplant mortality than the other group.However, albumin levels and the percentage of lymphocytes were lower.The ORs (95% CIs) for ACR and HRs (95% CIs) for mortality after LT based on the pre-transplantation serum vitamin D level are reported in Tables 5 and 6, respectively.In the crude and first adjusted model (based on age, sex, and BMI), there was a significant association between vitamin D deficiency in the pre-transplant time and odds of ACR (odds ratio [OR] = 2.71, 95% confidence interval [CI] 1.61-4.29,P for trend < 0.001; OR = 2.68, 95% CI 1.63-4.39,P for trend < 0.001, respectively).Furthermore, in model 2, after adjusting for further confounders (waiting time for transplantation, medical condition at the time of LT, causes of cirrhosis, smoke, alcohol, hypertension, diabetes mellitus, and infection of CMV) and final model (further adjustment for MELD-Na, AST, ALT, PMN, WBC, and lymphocytes), vitamin D deficiency was associated with 2.68-(OR: 2.68; 95% CI 1.59-4.71,P for trend = 0.001) and 2.69-fold (OR: 2.69; 95% CI 1.50-4.68,P for trend = 0.001) increase in the odds of ACR, respectively.
Although in the crude and first adjusted model, vitamin D deficiency in the pre-transplant stage was significantly associated with an increase in the risk of mortality after two years follow-up in cirrhotic patients undergoing transplantation (HR = 2.64, 95% CI 1.42-4.33;P for trend = 0.022; HR = 2.02, 95% CI 1.04-3.93,P for trend = 0.038, respectively), in the second and final adjusted model, no significant relationship was observed (HR = 1.63, 95% CI 0.80-3.31,P for trend = 0.177; HR = 1.46, 95% CI 0.71-3.00;P for trend = 0.309, respectively).The relationship between the MELD-Na score and serum vitamin D levels in the pre-transplantation period is demonstrated in Fig. 1.The findings suggested an inverse relationship between these two.The correlation coefficient r was − 0.157 (P < 0.001; 95% CI − 0.210, − 0.104).

Discussion
The current retrospective cohort study investigated the association between vitamin D deficiency prior to LT with ACR and mortality in liver transplant recipients.The study's findings indicated a significant correlation between vitamin D deficiency and increased odds of ACR after LT.Our finding is consistent with the investigation conducted by Doi et al., which demonstrated an association between vitamin D deficiency and a greater incidence of ACR in the short-term following LT compared to individuals with adequate vitamin D levels 11 .Zhou et al.   www.nature.com/scientificreports/also reported that a high 25(OH)D level (> 25 ng/mL) in the pre-transplant period is significantly associated with a lower ACR within 30 days following LT 17 .Furthermore, another study by Bitetto et al. corroborated these findings, establishing an independent association between low serum 25-hydroxyvitamin D levels (< 5 ng/mL) and the occurrence of moderate to severe ACR episodes within two months following LT 26 .Generally, these findings suggest that maintaining adequate levels of vitamin D during the pre-transplantation period plays a pivotal role in minimizing the risk of ACR following transplantation.Aside from this, some research has shown that vitamin D supplementation is associated with a lower risk of ACR 11,17,27 .According to a study by Grant al., supplementation with cholecalciferol (vitamin D3) for 12 weeks after LT increased vitamin D levels and reduced ACR and infection rates 27 .Hence, vitamin D deficiency can potentially affect ACR, and its correlation cannot only explained by deterioration in liver function.Additionally, we found that patients with vitamin D deficiency had higher scores on the MELD-Na than those with sufficient levels of vitamin D. This negative correlation has also been reported by Doi et al. 11 .MELD-Na/ MELD scores are widely used to assess liver disease severity and predict mortality in individuals with cirrhosis 28 .Thus, vitamin D deficiency may be associated with the severity of cirrhosis and adversely affect LT outcomes.Nevertheless, it should be noted that malnutrition is common among patients with ESLD 29 , and it is well documented that malnourished ESLD patients exhibit a high prevalence of vitamin D deficiency 20,21,30 .Malnutrition per se can have a negative effect on survival 31,32 .Hence, serum 25-hydroxyvitamin D levels may function as a prognostic marker for morbidity and mortality.
Our retrospective cohort study also demonstrated a significant correlation between pre-transplantation vitamin D deficiency and an elevated mortality risk after transplantation in both the crude and first-adjusted models.Nevertheless, this association was not statistically significant in the second and final adjusted models, suggesting that other potential confounding variables may impact the relationship between vitamin D deficiency and mortality in cirrhotic patients undergoing transplantation.Similarly, Doi et al. reported no significant disparity in overall survival between individuals with vitamin D deficiency and those with sufficient vitamin D levels before LT.However, a notable discrepancy was observed in post-transplantation periods, suggesting that vitamin D supplementation or maintaining adequate vitamin D levels following transplantation may be of benefit 11 .In addition, it has been shown that vitamin D supplementation during the first month after LT is associated with a better survival rate than the control group 17 .
Several mechanisms may explain the protective effect of vitamin D, including decreased production of inflammatory mediators such as interleukin (IL)-2, IL-17, and interferon-gamma (IFN-γ) [33][34][35] , decreased dendritic cells (DCs) maturation 36 , and an increase in Treg cell immunoprotective activity 37 .Treg are immune-suppressive cells that suppress the immune system, maintain self-tolerance, and prevent autoimmune diseases 38 .Vitamin D has been shown to manipulate monocytes and dendritic cells at different levels, allowing these cells to exert tolerogenic effects 39 .Additionally, vitamin D can inhibit macrophage transition to the M1 phenotype and promote macrophages with M2 phenotypes 40 .M1 macrophages secrete inflammatory cytokines that hinder cell proliferation, potentially resulting in tissue damage, while M2 macrophages facilitate cell proliferation and tissue regeneration 41 .Furthermore, vitamin D is thought to inhibit the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway and cyclooxygenase (COX)-2 transcription 42 , leading to a reduction in reactive oxidative species (ROS) that can cause oxidative stress 43 , DNA damage, and cellular death.
Moreover, research has suggested that bacterial infections may serve as a possible trigger for transplant organ rejection 44 .Vitamin D is known to preserve the integrity of the intestinal barrier by upholding the expression of immune cells within the tight junction, which is crucial for impeding bacterial invasions 45 .Besides, vitamin D receptors are also present within the gut barrier, promoting the production of antimicrobial peptides (AMPs) 46 and Claudin-5 expression 45 .The AMPs play a critical role in protecting against infection and innate immunity 47 .In addition to their broad-spectrum antimicrobial activity, these peptides possess diverse mechanisms of action and regulate the composition of the gut microbiome 48 .Claudin-5, a tight junction protein, plays a key role in maintaining the integrity of the bowel's physical barrier, and its disruption is directly related to intestinal inflammation 49 .
The present retrospective cohort study was conducted in two referral centers, which is a notable strength of the study.Additionally, the study had a relatively large sample size and included a comprehensive assessment of potential confounders while adjusting for multiple models, which further strengthens our findings.However, it is important to consider several limitations when interpreting the results.First, the study's retrospective design limits its ability to establish a causal relationship between vitamin D deficiency and ACR or mortality.Second, the study did not consider the effects of vitamin D supplementation, which may have affected the findings.Third, the characteristics of the donors were not included in the analysis, which could have impacted the results.Finally, we were not able to include immunosuppressant doses, which are used after transplantation, in our analysis.

Conclusion
The present study provides evidence that pre-transplant serum vitamin D levels may predict ACR in patients with cirrhosis undergoing LT.The findings of this study may have important implications for clinical practice, and further research is needed to fully understand the potential benefits and risks of vitamin D supplementation in these patients.

Table 1 .
Characteristics of all population study prior to liver transplantation.

Table 2 .
Characteristics of vitamin-D-deficient and vitamin D-sufficient patients prior to liver transplantation.a Obtained from Independent sample T-Test for continuous variables and Chi-square for Categorical variables.
PMN: polymorphonuclear neutrophil.Significant values are in bold.Groups,

Table 4 .
Characteristics of cirrhotic patients based on mortality status after liver transplantation.a Obtained from Independent sample T-Test for continuous variables and Chi-square for Categorical variables.BMI: Groups,

Table 5 .
Odds ratio (OR) and 95% confidence interval (CI) for acute cellular rejection (ACR) of liver transplantation (LT) based on the serum vitamin D level of pre-transplantation. **Binary logistic regression was used to obtain OR and 95% CI. *Model 1: adjusted for age, sex, and BMI.† Model 2: Model 1 + adjusted for waiting time for transplantation, medical condition at the time of LT, causes of cirrhosis, smoke, alcohol, hypertension, diabetes mellitus, and infection of CMV.‡ Model 3: Model 2 + MELD-Na, AST, ALT, PMN, WBC, and lymphocytes.Significant values are in bold.

Table 6 .
Hazard ratio (HR) and 95% confidence interval (CI) for mortality after liver transplantation (LT) based on the serum vitamin D level of pre-transplantation. **Cox regression was used to obtain HR and 95% CI. *Model 1: adjusted for age, sex, and BMI.† Model 2: Model 1 + adjusted for waiting time for transplantation, medical condition at the time of LT, causes of cirrhosis, smoke, alcohol, hypertension, diabetes mellitus, and infection of CMV.‡ Model 3: Model 2 + MELD-Na, AST, ALT, PMN, WBC, and lymphocytes.Significant values are in bold.