Effect of off-label vitamin D analog use for albuminuria in early nondiabetic nephropathy: a double-blind, randomized, placebo-controlled trial
Article information
Abstract
Background
Albuminuria is one of the factors promoting the progression of chronic kidney disease (CKD). The study aimed to assess the efficacy and safety of calcitriol for the reduction of microalbuminuria in patients with nondiabetic nephropathy.
Methods
In this randomized, double-blind, placebo-controlled, and multicenter study, adult patients with nondiabetic CKD stage 3 or greater and albuminuria were included. Participants were administered calcitriol or placebo for 6 months and followed for up to 12 months. The primary outcome was the change in urine protein-to-creatinine ratio (UPCR), and secondary outcomes included the changes in renal function and vitamin D level. The safety was assessed by recording adverse events during the treatment and follow-up.
Results
A total of 159 subjects were enrolled. The UPCR at 24 and 48 weeks was significantly decreased compared to the baseline in the calcitriol group (ΔUPCR, –0.24 g/g [95% CI, –0.43 to –0.05] and –0.22 g/g [95% CI, –0.43 to –0.01], respectively), but the mean changes of UPCR during 24 weeks and 48 weeks were no significant difference between the two groups. No significant differences were in the change in renal function and vitamin D level. Seventy-eight adverse events were reported during the treatment phase, and there were no significant differences in the type or frequency of adverse events between the two groups.
Conclusion
Although calcitriol treatment showed a significant reduction of proteinuria from baseline, the effect was insufficient in nondiabetic CKD compared to placebo. Therefore, the use of calcitriol for the reduction of albuminuria is worth considering.
Introduction
The presence of proteinuria is considered a risk predictor of chronic kidney disease (CKD). Several studies have demonstrated that high levels of urinary protein (urine protein-to-creatinine ratio [UPCR], >0.2 g/g) are closely related to both disease progression and cardiovascular outcomes [1–3]. Since albuminuria is reversible in the early stages of kidney damage, it should be identified in high-risk patients, and treatment modalities to reduce proteinuria may delay the onset of CKD.
Angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB) have been shown to reduce proteinuria more effectively than other antihypertensives, which may result from alterations in glomerular hemodynamics and renin-angiotensin system (RAS) inhibition [4,5]. However, in patients with normal blood pressure, since the use of RAS blockers may have negative effects such as hypotension, they still suffer from proteinuria. Therefore, studies were conducted to find various drugs to reduce persistent proteinuria despite the use of ACEi or ARB, such as sulodexide [6], pentoxifylline [7], and vitamin D analogs [8].
There are several hypotheses for how vitamin D might reverse the progression of kidney disease and reduce fibrosis [9]. Activated vitamin D inhibits renin biosynthesis and the endocrine regulation of hyperglycemia by the RAS [10–12]. Emerging evidence in patients with CKD has shown that vitamin D can have a renoprotective effect by reducing proteinuria or albuminuria, even in diabetic nephropathy [13,14]. Other studies conducted in patients with immunoglobulin A (IgA) nephropathy have reported that active vitamin D reduces proteinuria [8,15]. However, this beneficial effect in nondiabetic nephropathy has not yet been clinically demonstrated, and the results of clinical studies estimating the effect of vitamin D on proteinuria have been inconclusive.
Therefore, we conducted this randomized trial to prospectively assess the effectiveness of an active vitamin D analog for the reduction of microalbuminuria in patients with nondiabetic nephropathy. Additionally, we studied the persistent effects of vitamin D treatment on albuminuria, renal function, and blood pressure.
Methods
Study design and participants
This multicenter, randomized, double-blind study was performed between October 2011 and December 2014. Patients 19 years and older with nondiabetic nephropathy defined by an estimated glomerular filtration rate (eGFR) of ≥30 mL/min/1.73 m2 and UPCR at first-morning void of >0.2 g/g, serum parathyroid hormone concentration of <500 pg/mL, and serum calcium concentration of less than 10.2 mg/dL and receiving a stable dose of an ACEi and/or ARB for 3 months or more with optimal blood pressure control (systolic/diastolic blood pressure of ≤140/90 mmHg) were eligible for inclusion. Patients with normotension were included even if they did not use ACEi or ARB. Exclusion criteria were diabetes mellitus, uncontrolled hypertension, nephrotic-range proteinuria (urine protein, >3.5 g/day), rapid progressive glomerulonephritis, end-stage renal disease requiring renal replacement therapy, New York Heart Association class III–IV heart failure or left ventricular ejection fraction <40% in the previous 6 months, severe chronic obstructive pulmonary disease history, decompensated liver disease, active malignancy with a life expectancy less than 10 years, treatment with vitamin D analog in the previous 3 months, hypersensitivity to vitamin D analog, use of immunosuppressive treatment, noncompliance with the study medication, and no signed informed consent.
The study protocol was approved by an independent ethics committee of the central and two local institutions (No. 1109-080-378, 10-2012-007, and 12-2012-0009), and all patients provided written informed consent. This trial is registered with ClinicalTrials.gov with the number of NCT01512862.
Randomization and masking
The randomization sequence was computer generated by the statistician, based on permuted block sizes of four, and preserved in a sealed envelope to conceal allocation. Patients were randomly assigned in a 1:1 ratio to receive fixed-dose calcitriol (Calcio; Hanmi) daily or a matched placebo. Study drugs and packaging were identical in appearance. The study investigator, participants, and statistical analyzer were masked to treatment assignment for the duration of the study.
Study treatment and procedures
The study consisted of two phases: the treatment phase and the follow-up phase (Supplementary Fig. 1, available online). Patients in all groups received one capsule daily of calcitriol (0.25 μg) or matched placebo during the first 6 weeks, and the dose was titrated up to two capsules daily at the second visit if they did not experience any adverse events. For 24 weeks (treatment phase), patients who had adverse reactions were again reduced to one capsule of the study drug. Doses of ACEi or ARB could not be adjusted after randomization. They could be given an antihypertensive drug to control blood pressure (<130/80 mmHg), following the KDIGO (Kidney Disease: Improving Global Outcomes) guideline. The addition of other drugs, except steroids and/or immunosuppressants, was avoided if necessary for clinical situations, such as the addition of medications known to affect the contraindications or to reduce proteinuria (e.g., pentoxifylline, sulodexide, etc.). In addition, all participants were instructed to have a low-salt diet during the treatment phase according to the previous study results [16].
Patients were examined at baseline, 6 weeks, 12 weeks, and 6 months after randomization during the treatment phase, and at every 3 months after treatment completion (follow-up phase). At baseline, demographic information was collected. Blood pressure and pulse, adverse events, concomitant drugs, and adherence to study drugs were assessed at every visit. Blood chemistry including serum creatinine, calcium, phosphate, albumin, and urine specimens gathered at first-morning void were measured at baseline, 12, 24, 36, and 48 weeks of treatment. In addition, plasma concentrations of vitamin D [25-hydroxyvitamin D3 (25(OH)D3)] were also obtained at baseline, 12, 24, 36, and 48 weeks of treatment.
Outcomes
The primary efficacy outcome was the mean change in UPCR from baseline to the last measurement during the treatment phase. Secondary efficacy outcomes were the changes in the mean UPCR and urine albumin-to-creatinine ratio (UACR) between the baseline and last study evaluation (48 weeks) and the proportion of patients achieving at least a 15% reduction in proteinuria. Additional efficacy outcomes included mean change in eGFR during the treatment and follow-up phases and the percentage changes in renal damage markers from baseline to the last measurement during the treatment phase. Absolute proteinuria changes were calculated using the last measurement of UPCR minus the baseline measurement of UPCR. The percentage change of proteinuria was calculated by dividing the change in absolute UPCR by the baseline measurement of UPCR. The eGFR was calculated by using the MDRD (Modification of Diet in Renal Disease) equation. Safety outcomes included adverse events reported by patients and changes in laboratory values. All adverse events were classified and confirmed by an independent researcher who was masked to treatment assignment. Drug compliance was measured at every visit to all patients.
Statistical analysis
The intention-to-treat analysis included all randomized patients with at least one dose of the study drug and was used for all efficacy and safety analyses. Patients with missing data on changes in efficacy and safety measures between two time points were excluded from analyses because the change value could not be calculated. Continuous variables are reported as mean ± standard deviation. Categorical variables are reported in terms of frequency and percentage. For normally distributed variables, the difference in mean change between the two groups was analyzed by the unpaired t test. The differences in mean changes within groups were analyzed by the paired t test. For nonnormally distributed variables, the Mann-Whitney U test was used to analyze differences between the two groups and within groups. Categorical outcomes, including the incidence of adverse events, were analyzed by the chi-square or Fisher exact test. All statistical analyses were performed using IBM SPSS version 23 (IBM Corp.).
We calculated that a total sample size of 94 patients (47 patients per arm) was needed for at least 80% power to detect an average percentage difference in UPCR of 40% between the two groups at a two-sided significance level of 0.05. A 40% dropout rate was considered, and a total of 156 patients were needed.
Results
A total of 159 subjects from the three institutions were enrolled and randomly assigned to calcitriol (n = 79) or placebo (n = 80) (Supplementary Fig. 2, available online). A total of 135 subjects completed the study medication requirements during the treatment phase and entered the follow-up phase. During the follow-up phase, eight subjects dropped out. Finally, 67 subjects in the calcitriol group and 60 subjects in the control group completed the study. Of the 32 subjects who dropped out, one subject in the calcitriol group and four subjects in the placebo group stopped participating in the study because of side effects, such as mild headache, hypotension, and slight edema. Another 15 subjects withdrew their voluntary consent, and five were lost to follow-up during the study period. Participant demographics are depicted in Table 1. Baseline characteristics, clinical, and biochemical information, comorbid disease, and concomitant medications were balanced between the calcitriol and placebo groups. In both groups, more than 70% of the participants were hypertensive and most of them were using ACEi or ARB to treat hypertension. Five subjects in the calcitriol group and 12 subjects in the placebo group were treated with other drugs for proteinuria such as pentoxifylline and sulodexide. The mean 25(OH) vitamin D3 concentrations were 17.16 ± 6.21 ng/mL and 18.01 ± 9.26 ng/mL in the calcitriol and placebo groups, respectively, which indicated vitamin D deficiency status.
Patient demographics at baseline
Efficacy outcomes
Changes in proteinuria
In the intention-to-treat analysis, the UPCR at 24 and 48 weeks were significantly decreased compared to baseline only in the calcitriol group, not in the placebo group (calcitriol vs. placebo: ΔUPCR at 24 weeks, –0.24 ± 0.79 g/g [95% CI, –0.43 to –0.05] vs. –0.17 ± 0.08 g/g [95% CI, –0.33 to 0]; at 48 weeks, –0.22 ± 0.85 g/g [95% CI, –0.43 to –0.01] vs. –0.06 ± 0.65 g/g [95% CI, –0.23 to 0.11]) (Table 2). In addition, the UACR at 24 weeks was significantly decreased in the calcitriol and placebo groups. However, the 24-week proteinuria change was analyzed using repeated measures analysis, the mean changes in UPCR and UACR during 24 weeks were not statistically significant between the two groups (ΔUPCR, p = 0.50; ΔUACR, p = 0.85). The changes in albuminuria and proteinuria with adjustments for changes in body mass index, mean arterial pressure, cause of CKD, and comorbid hypertension were not significantly different between the two groups. UACR and UPCR were increased during the follow-up phase after the end of administration. In the comparison of the UPCR change over 48 weeks, there was a significant decrease compared to the baseline in the calcitriol group (p = 0.04), but there was no difference from the baseline in the placebo group (p = 0.56). However, there was no statistically significant difference in the level of UPCR at 48 weeks between the two groups (calcitriol vs. placebo, 0.82 ± 0.68 g/g vs. 0.83 ± 0.72 g/g; p = 0.89). The proportion of patients achieving at least a 15% reduction in proteinuria was not different between the calcitriol and placebo groups (38 and 36 subjects, respectively) (p = 0.70).
Comparison of changes in proteinuria from baseline to 24 weeks and 48 weeks between the two groups by repeated measured analysis
Changes in renal function, vitamin D, and blood pressure
As secondary outcomes, the changes in renal function, serum vitamin D3 level, and mean blood pressure were estimated and the differences between the two groups were analyzed at 24 weeks and 48 weeks (Fig. 1). As an evaluation of the effect of calcitriol on delaying the progression of kidney disease, serum creatinine concentration and eGFR were not changed significantly during either the treatment phase or the follow-up phase. The level of 25(OH) vitamin D3 declined during the treatment phase in the calcitriol group, whereas the level increased in the placebo group. However, there was no statistically significant difference.
Effect of calcitriol on SCr, eGFR, 25(OH) vitamin D3, and MAP in the calcitriol-treated group (●) or placebo group (▲).
(A) Changes in SCr during 48 weeks in the intention-to-treat analysis. (B) Changes in estimated glomerular filtration rate (eGFR; calculated using IDMS MDRD equation) during 48 weeks in the intention-to-treat analysis. (C) Changes in 25(OH) vitamin D3 during 48 weeks in the intention-to-treat analysis. (D) Changes in MAP during 48 weeks in the intention-to-treat analysis.
IDMS MDRD, Isotope Dilution Mass Spectrometry-Modification of Diet in Renal Disease; MAP, mean arterial pressure; SCr, serum creatinine.
Compliance
Compliance with the treatment intervention in the calcitriol and placebo group was similar in the treatment phase (calcitriol vs. placebo: 94.66% ± 5.98% vs. 92.67% ± 10.20%, respectively). The number of subjects with low compliance (less than 70%) was four in the placebo group and 0 in the calcitriol group. When the compliance was corrected, there was no significant difference in the change in UACR or UPCR between the two groups.
Per-protocol analysis
In the per-protocol analysis, both UACR and UPCR were significantly decreased in the calcitriol group during the treatment phase, whereas an insignificant decrease was observed in the placebo group (calcitriol vs. placebo: ΔUACR, –0.12 g/g [95% CI, –0.22 to –0.03] vs. –0.09 g/g [95% CI, –0.19 to 0.002]; ΔUPCR, –0.26 g/g [95% CI, –0.46 to –0.06] vs. –0.15 g/g [95% CI, –0.32 to 0.02]) (Supplementary Table 1, available online).
Safety outcomes
A total of 78 adverse events were reported during the treatment phase following the administration of calcitriol and placebo (Table 3). There were no statistically significant differences in the type or frequency of adverse events between the two groups. The most commonly reported adverse events in the calcitriol and placebo groups were upper respiratory tract infections and gastrointestinal side effects. Urinary discomfort, hyperuricemia, myalgia, and headache were also reported in the calcitriol group, and some patients in the placebo group had hyperlipidemia, edema, hyperuricemia, and hypotension. Among the 40 adverse events recorded in the calcitriol group, 30 cases (75.0%) were classified as mild, and 10 cases (25.0%) as moderate. Furthermore, 35 cases (87.5%) of the adverse events in the calcitriol group were assessed unlikely to be related to the calcitriol, with three cases (7.5%) considered possibly related, including two cases of constipation and one case of headache. Within the placebo group, 33 mild (86.8%) and five moderate events (13.2%) were reported, with the majority (92.1%) assessed as unlikely to be related to the administered drug.
Adverse events in the treatment phase (during 24 weeks)
Subgroup analysis
In the subgroup analysis of IgA nephropathy patients (n = 69), there was no significant difference in the change in proteinuria, blood pressure, or renal function between the two groups (Supplementary Table 2, available online). In addition, primary and secondary outcomes did not differ significantly between the two groups among patients taking RAS blockers or proteinuria-lowering agents (Supplementary Table 3, available online). Furthermore, in the subgroup analysis for 32 patients who were not using renin-angiotensin-aldosterone system (RAAS) blockers, with 17 in group 1 and 15 in group 2, there was no significant difference in the change in UPCR between the two groups at 6 months and 12 months.
Discussion
In this study, calcitriol administration significantly reduced proteinuria and albuminuria compared to pretreatment. However, we found no evidence of reduced proteinuria compared to placebo. In addition, no significant results were obtained on improving renal function as an expected effect of proteinuria reduction. In terms of safety, no significant abnormalities were found compared with placebo, and most of them were mild and transient.
The purpose of this study was to evaluate the effect of reducing proteinuria with calcitriol, because there are few treatment options available except RAS blockers [17], even though proteinuria is a major factor in the progression of kidney disease [18]. Especially in patients with normal blood pressure, RAS blockers should be used with caution because there is a risk of causing hypotension or hyperkalemia. For this reason, interest in vitamin D analogs as a proteinuria-reducing agent has been increased, and it is being used as an off-label indication for clinical use.
The mechanism by which vitamin D reduces proteinuria is associated with anti-inflammatory action. Several studies have already suggested a potential anti-inflammatory mechanism of vitamin D in CKD. In vitro studies have attenuated the expression of tumor necrosis factor-alpha–induced monocyte chemoattractant protein 1 in proximal tubule cells [19]. Transforming growth factor-beta was inhibited in mesangial and juxtaglomerular cells. Paricalcitol, a synthetic vitamin D receptor (VDR) blocker, inhibits VDR and mediates nuclear factor kappa-B signaling, which inhibits inflammation in the podocyte system of the kidney [20]. In addition, in animal models of primary glomerular diseases, administration of vitamin D reduces albuminuria, improves glomerulosclerosis, and improves tubular function by reducing glomerular infiltration of inflammatory cells [21–23]. Based on the evidence from these in vitro and in vivo studies, decreased inflammation was observed in early CKD patients with higher serum vitamin D levels [24]. Since inflammatory responses increase vascular permeability [25] and glomerular vascular damage eventually results in proteinuria [26], modulating inflammatory responses is associated with reducing proteinuria. In another mechanism, vitamin D influences renin and angiotensin II levels by negatively regulating the renin gene. Li et al. [10] showed that renin expression and plasma angiotensin II production were increased several-fold in VDR-null mice compared to wild-type mice. Park et al. [27] also reported that intervention with calcitriol decreased plasma renin and angiotensin II levels in hemodialysis patients with secondary hyperparathyroidism. According to this mechanism, the combined use of RAAS blockers and vitamin D may better reduce proteinuria.
In clinical practice, the antiproteinuric effect of vitamin D has been reported in various patient groups, including diabetic kidney disease and IgA nephropathy. In one study of patients with diabetic nephropathy (UACR, >0.03 g/g), UACR was significantly decreased in the treatment and control groups after administration of vitamin D at 50,000 IU/week, and there was a significant difference between the groups (p = 0.028) [28]. In another study by Liyanage et al. [29], high-dose vitamin D (50,000 IU/week) was also associated with a significant reduction in UPCR compared to distilled water (p = 0.001). The eGFR was significantly increased in the vitamin D-administered group only (p = 0.03 for the between-group difference). One of the reasons for the discrepancy between our findings and theirs is that the patient groups were different. In patients with diabetic nephropathy, there may be more glomerular vascular injury due to hyperglycemia and more proteinuria, which may result in a greater antiproteinuric effect of calcitriol. Another reason is the different doses of vitamin D used in clinical trials. In the previous study, when high-dose vitamin D was injected intramuscularly at 50,000 IU/week, a renoprotective effect was shown in CKD patients [30]. The dose of the study is equivalent to intravenous administration of 1,050 μg/week in terms of its bioavailability. Since the weekly dose administered in our study was 2.45 μg/week (0.5 μg/day for 7 days and given 70% bioavailability [31]), the dose in those two studies was 400 times higher than in our study. In addition, the bioavailability of vitamin D is reduced in CKD patients. Vitamin D-binding protein (DBP) excretion is promoted and DBP reabsorption through the proximal tubule is inhibited in patients with proteinuria, leading to a reduction of DBP [32,33]. In patients with low DBP levels, the clearance of active vitamin D may have been enhanced, resulting in less efficacy of calcitriol to decrease proteinuria.
Additionally, several studies were conducted in nondiabetic CKD patients. In patients with IgA nephropathy, Liu et al. [15] used a calcitriol dose similar to ours in IgA nephropathy patients with persistent proteinuria of protein excretion >0.8 g/day. After 48 weeks of 0.5 μg calcitriol twice weekly, the UPCR was significantly reduced compared to that of the control group (p = 0.03). Although the study showed a decrease in proteinuria, calcitriol did not affect recovery of renal function or changes in blood pressure. Despite the small number of patients (n = 50), the effect of reducing UPCR appears to be due to the longer treatment duration of 48 weeks in patients with a single disease. A meta-analysis of seven randomized controlled trials (RCTs; n = 310) also reported the antiproteinuric effects of calcitriol in patients with IgA nephropathy [8]. In that study, calcitriol contributed to a significant decrease in proteinuria, but no significant differences were observed in the change in serum creatinine, serum calcium, or serum phosphorus level. Most of the studies have been conducted in patients with IgA nephropathy, so the evidence is limited in their application to other nondiabetic patients. Another RCT, conducted in nondiabetic stage III–IV CKD patients, reported that paricalcitol did not alter plasma levels of renin and UACR [34]. The duration of treatment was shorter than in our study, but the treatment dose (2 µg) was equivalent to 0.5 µg/day in our study. However, there is still insufficient evidence to demonstrate the effect of calcitriol on reducing albuminuria in nondiabetic patients. In this regard, our results are meaningful in that the effects of calcitriol were evaluated in various nondiabetic patients.
Nevertheless, there were several limitations to this clinical trial. In particular, unlike other studies, the antiproteinuric effect of calcitriol could not be determined because proteinuria was significantly reduced in the placebo group. This can be explained as follows: first, the subjects included in this study had microalbuminuria, which is a relatively small amount of albumin in the urine. Since the probability of diagnosing albuminuria in microalbuminuric patients (UACR, ≥0.03 g/g) was 47.2% based on a dipstick analysis [35], it was difficult to define whether the test results in the participants were an actual increase or decrease in albuminuria or errors in the test. In particular, we tried to collect samples of the first urine in the morning to measure proteinuria, but the urinalysis time of patients varied. In addition, our ability to measure the exact effect of the test drug was limited because the protein in the urine was diluted according to the patient’s water or sodium intake. Second, we recommended that the participants have a low-salt diet, but the amount of sodium intake per day was not evaluated. Many prior studies have reported that low-salt diets significantly affect proteinuria reduction. Hwang et al. [36] reported that the amount of daily albuminuria was significantly decreased in the intensive low-salt diet education group compared to the conventional education group (p < 0.001). In the Keyzer et al. study [37], paricalcitol had no significant effect on albuminuria in patients, but low-salt diets significantly reduced albuminuria and decreased urinary excretion of sodium in patients with ACEi (p < 0.001). It seems that there was a difference in the treatment effect according to compliance with the low-salt diet in our study, but we could not consider the diet effect when assessing the changes in proteinuria. The urinary sodium excretion is closely correlated with dietary salt intake; therefore, it would be necessary to consider changes in urinary sodium concentration as a confounding factor in the relationship between vitamin D and proteinuria in further studies. Finally, the short treatment period and follow-up period did not allow us to determine the long-term antiproteinuric effect of vitamin D, especially on the recovery of renal function. In this study, both calcitriol and placebo reduced proteinuria over 6 months, but a sustained effect on proteinuria was observed only in the calcitriol group. Therefore, calcitriol’s effect on reducing proteinuria can be measured more clearly under long-term administration. A long-term study is needed to evaluate the reduction of proteinuria and recovering renal function after dosing longer than 12 months.
In conclusion, while a significant reduction in proteinuria was observed following calcitriol use without notable adverse events, no significant difference in proteinuria reduction was found between the calcitriol and the placebo. Therefore, calcitriol treatment for reducing albuminuria requires careful consideration due to insufficient evidence. Further real-world studies are needed to evaluate the antiproteinuric effects considering confounding factors.
Supplementary Materials
Supplementary data are available at Kidney Research and Clinical Practice online (https://doi.org/10.23876/j.krcp.24.051).
Notes
Conflicts of interest
The study was investigator-initiated and after an application, Hanmi provided the investigational drugs and placebo. However, Hanmi has not participated in intellectual design, in any of the practical parts, analysis, or manuscript preparation. Nn, YSK, and JMO had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have no other conflicts of interest to declare.
Funding
The data presented in this study are available from the corresponding author upon reasonable request.
Data sharing statement
The data presented in this study are available from the corresponding author upon reasonable request
Authors’ contributions
Conceptualization: NH, YSK, JMO
Data curation: NH, DKK, HL, KWJ, SK, JPL
Formal analysis: NH, DKK, HL
Funding acquisition: JMO
Project administration: YSK, JMO
Writing–original draft preparation: NH
Writing–review & editing: All authors
All authors read and approved the final manuscript.