The authors have declared that no competing interests exist.
Conceived and designed the experiments: HC YB WJ. Performed the experiments: YH JL HY KG LZ. Analyzed the data: YH HC HY. Contributed reagents/materials/analysis tools: JL. Wrote the paper: YH HC.
Low vitamin D levels can be associated with albuminuria, and vitamin D analogs are effective anti-proteinuric agents. The aim of this study was to investigate differences in vitamin D levels between those with micro- and those with macroalbuminuria, and to determine whether low dose cholecalciferol increases vitamin D levels and ameliorates albuminuria.
Two studies were performed in which 25-OH vitamin D3 (25(OH)D3) concentrations were determined by electrochemiluminescence immunoassay: 1) a cross-sectional study of patients with type 2 diabetes mellitus (T2DM) (n = 481) and healthy controls (n = 78); and 2) a longitudinal study of T2DM patients with albuminuria treated with conventional doses, 800 IU, of cholecalciferol for 6 months (n = 22), and a control group (n = 24).
1) Cross-sectional study: Compared to controls and T2DM patients with normoalbuminuria, serum 25(OH)D3 concentrations were significantly lower in patients with macro-albuminuria, but not in those with micro-albuminuria. Serum 25(OH)D3 levels were independently correlated with microalbuminuria. 2) Longitudinal study: Cholecalciferol significantly decreased microalbuminuria in the early stages of treatment, in conjunction with an increase in serum 25(OH)D3 levels.
Low vitamin D levels are common in type 2 diabetic patients with albuminuria, particularly in patients with macroalbuminuria, but not in those with microalbuminuria. Conventional doses of cholecalciferol may have antiproteinuric effects on Chinese type 2 diabetic patients with nephropathy.
Diabetes has become a major public health problem in China, and a large-scale epidemiological survey revealed a prevalence of 9.7% approximately 5 years ago
Epidemiological studies have shown that low 25(OH)D3 levels are common in patients with albuminuria (spot urinary albumin/creatinine ratio (ACR) ≥30 mg/g)
Albuminuria can be divided into two phases, micro- and macro-albuminuria. Patients with macroalbuminuria which is a more serious stage of DN, has a poor prognosis. Whether serum 25(OH)D3 levels can indicate the severity of DN in patients with micro and macro-albuminuria hasn’t been reported yet. Since high dose and long-term of vitamin D administration may cause a variety of side effects such as electrolyte imbalance problem, whether regular doses of vitamin D3 can improve proteinuria level either. There is no report about them. Thus this study is to observe the differences in serum 25(OH)D3 levels between patients with microalbuminuria and macroalbuminuria, and to investigate the effects of nutritional vitamin D supplementation with low dose of cholecalciferol, in addition to RAAS inhibitors, in Chinese patients with DN.
All subjects gave written informed consent, and the study was approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and complied with the Declaration of Helsinki.
The protocol included a cross-sectional study and an open-label longitudinal study.
All patients in both studies were recruited from the outpatient clinic at the Shanghai Clinical Center for Diabetes (Shanghai, China) from January, 2011 to April, 2012. At the same time, seventy-eight healthy patients were enrolled as normo-glycemic control subjects without any history of kidney diseases or current urinary tract infection and have no supplements with vitamin D, active vitamin D analogs, or any steroid. Patients with diabetes were categorized as those with normo-albuminuria (NA), when the ACR was persistently <30 mg/g (n = 261), those with microalbuminuria (MA), when the ACR was between 30 and 300 mg/g (n = 154), and those with DN, if they had persistent albuminuria (>300 mg/g) (n = 66), without any other kidney or renal tract disease. Among them, patients were eligible if they were older than 20 years, with T2DM and ACR persistently >30 mg/g, and on stable doses of ACE inhibitors or angiotensin receptor blockers (ARBs) for 3 months or more, without supplement with vitamin D, active vitamin D analogs, or any steroid, serum parathyroid hormone concentration of 25–500 ng/L and serum calcium concentration of less than 2.45 mmol/L were enrolled into the longitudinal intervention study
Demographic and clinical data, including age, sex, duration of diabetes, weight, height, and medication, were recorded. Blood pressure (BP) was measured twice with a Hawksley sphygmomanometer after 10 minutes of supine rest. The ACR was determined in three consecutive spot urine samples using the Dade Behring Nephelometer II System (antiserum to human albumin, Siemens Healthcare Diagnostics). The estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease study equation (MDRD)
Serum samples were maintained at −70°C for subsequent assays. Serum 25(OH)D3 measurements were performed using a commercially available electrochemiluminescence immunoassay (ECLIA) (Roche Diagnostics GmbH), according to the manufacturer’s protocol. The detection limit of human serum 25(OH)D3 assay was 4 ng/ml. Duplicate measurements were obtained for all samples.
Each variable was assessed for normal distribution. Data are expressed as mean ± standard deviation (SD) for normally distributed variables, and as median with the interquartile range for skewed variables. Skewed variables were natural logarithm-transformed to improve normality prior to analysis, and then re-transformed to their natural units in order to present them in a tabulated form. Characteristics of subjects across different patient groups were compared by ANOVA and analysis of covariance, and those between control and patient groups were compared using the t-test. Comparisons between groups before and after cholecalciferol treatment were undertaken with the Wilcoxon signed-ranked test. Pearson correlation tests, multivariable linear regression analyses, and partial correlation analyses were also performed. Only variables that were significantly (P<0.05) related to 25(OH)D3 by Pearson correlation analyses were entered into the multiple linear stepwise regression analysis. All calculations were undertaken using GraphPad Prism software (GraphPad; San Diego, CA) and the statistical package for social sciences (SPSS) 17.0 software (Los Angeles, CA). All reported P-values were 2-tailed, and P-values <0.05 were considered statistically significant.
General characteristics and clinical parameters of the cross-sectional study are summarized in
Type 2 diabetes | ||||
Control subjects | Normoalbuminuria | Microalbuminuria | Macroalbuminuria | |
Case (M/F) | 78 (29/49) | 261 (171/90) | 154 (87/67) | 66 (44/22) |
Age (years) | 51.62±13.10 | 54.61±15.47 | 61.36±11.47 |
59.31±11.76 |
SBP(mmHg) | 129±18 | 131±17 | 141±23 |
146±21 |
DBP(mmHg) | 80±11 | 80±10 | 82±11 | 86±10 |
BMI(Kg/m2) | 23.76±2.58 | 24.24±3.36 | 24.99±3.7 |
26.28±4.3 |
HbA1c(%) | 5.30±0.25 | 8. 40±2.00 |
8.42±1.92 |
8.42±1.94 |
FPG(mmol/l) | 5.01±0.34 | 8.65±3.43 |
8.51±3.89 |
8.38±2.90 |
PG2H(mmol/l) | 6.14±1.02 | 13.16±4.66 |
12.88±4.47 |
13.40±5.66 |
Antidiabetic drugs n(%) | ||||
Monotherapy | 163 (62.5%) | 80 (51.9%) | 16 (24.2%) | |
Two-drugs | 60 (23.0%) | 46 (29.9%) | 13 (19.7%) | |
≥Three-drugs | 38 (14.5%) | 28 (18.2%) | 37 (56.1%) | |
TC(mmol/l) | 4.97±0.84 | 4.88±1.02 | 4.90±1.07 | 5.55±1.46 |
LDL-c(mmol/l) | 3.32±0.83 | 2.97±0.86 |
2.99±0.95 |
3.16±1.18 |
TG(mmol/l) | 1.57±1.01 | 1.70±1.67 | 1.86±1.34 | 2.66±2.16 |
HDL-c(mmol/l) | 1.33±0.28 | 1.26±0.41 | 1.18±0.29 |
1.15±0.40 |
Antilipidemic drug | ||||
Statins | 16 (6.1%) | 24 (15.6%) | 14 (21.2%) | |
Fibrats | 2 (0.8%) | 6 (3.9%) | 4 (6.1%) | |
Cr(mmol/l) | 69.06±12.47 | 68.47±16.96 | 71.17±26.23 | 122.47±75.86 |
ACR(mg/g) | 6.74(4.82–10.71) | 7.65(5.06–12.02) | 71.22(42.16–127.43) | 1078.58(552.91–2489.17) |
MDRD | 116.66±25.31 | 122.16±43.29 | 118.43±44.64 | 76.22±40.14 |
Hypertention(%) | 23(29.5%) | 112(42.9%) | 97(63.0%) | 59(89.4%) |
Anti-hypertention medication | ||||
RAS inhibitor (+) | 49 (18.8%) | 85 (55.2%) | 46 (69.7%) | |
HOMA-IR | 1.42±0.57 | 1.73±0.81 | 2.30±4.58 |
2.45±1.36 |
Median 25(OH)D3 concentrations in our sample were significantly higher in men than in women (13.34 (9.28, 18.17) ng/ml vs. 11.62 (7.58, 15.81) ng/ml; P<0.01) (
Both in male and female, mean serum 25(OH)D3 levels were significantly lower than in the control group (11.37 (6.48, 14.06) ng/ml vs. 15.82 (13.13, 20.29) ng/ml) and 7.57 (4.00, 10.73) ng/ml vs. 11.79 (8.07, 16.52) ng/ml, P<0.05 and 0.05, respectively) (
To further investigate the relationship between DN and serum 25(OH)D3, we divided the study population into quartiles based on serum 25(OH)D3 concentrations (8.56, 12.50, 17.15 ng/mL), with quartile 1 representing individuals with the lowest levels of 25(OH)D3 (
As vitamin D levels were lower in patients with DN, we performed a correlation analysis which including all subjects to investigate related factors. The Pearson correlation analysis suggested that serum 25(OH)D3 levels were significantly correlated with age, BMI, fasting plasma glucose (FPG), high density lipoprotein-cholesterol (HDL-c), fasting C-peptide (OFCP), PTH and ACR in males, while in females, serum 25(OH)D3 levels were significantly correlated with age, glutamate transaminase (ALT), glutamic-oxal(o)acetic transaminase (AST), potassium (K ), Sodium (Na), phosphorus (Pi), PTH and ACR (
male | female | |||
variables | r | P | r | P |
Age | 0.187 | 0.001 |
0.204 | 0.002 |
BMI | 0.139 | 0.012 |
−0.018 | 0.787 |
SBP | 0.027 | 0.626 | −0.054 | 0.422 |
HbA1c | −0.108 | 0.059 | −0.036 | 0.599 |
OFG | −0.113 | 0.042 |
−0.039 | 0.556 |
TC | −0.038 | 0.494 | −0.031 | 0.640 |
TG | −0.001 | 0.979 | −0.029 | 0.668 |
HDL | −0.175 | 0.002 |
−0.010 | 0.879 |
LDL | 0.032 | 0.571 | −0.018 | 0.786 |
lnCRP | −0.063 | 0.263 | −0.048 | 0.473 |
ALT | −0.005 | 0.924 | 0.137 | 0.040 |
AST | −0.022 | 0.698 | 0.178 | 0.008 |
BUN | 0.053 | 0.344 | 0.006 | 0.928 |
Cr | −0.041 | 0.462 | −0.114 | 0.087 |
UA | 0.058 | 0.297 | 0.040 | 0.550 |
MDRD | −0.064 | 0.251 | 0.004 | 0.956 |
K | −0.059 | 0.335 | −0.192 | 0.014 |
Na | −0.046 | 0.475 | −0.171 | 0.048 |
Cl | 0.097 | 0.133 | −0.097 | 0.264 |
Ca | −0.072 | 0.242 | −0.150 | 0.058 |
Pi | 0.026 | 0.679 | −0.250 | 0.001 |
lnPTH | −0.168 | 0.002 |
−0.200 | 0.002 |
lnACR | −0.138 | 0.012 |
−0.186 | 0.005 |
lnHOMA-IR | −0.084 | 0.457 | −0.004 | 0.980 |
In order to elucidate independent relationships between vitamin D and clinical parameters, we selected 25(OH)D3 as a dependent variable and other clinical parameters as the independent variables, thereby building a multiple linear stepwise regression equation. Only variables that were significantly (P<0.05) related to 25(OH)D3 by Pearson correlation analyses were entered into the multiple linear stepwise regression analysis. The results revealed an independent correlation between 25(OH)D3 and uACR (β = −0.290, P<0.01) in men, and the other four independent parameters were age (β = 0.191, P = 0.001), BMI (β = −0.168, P = 0.007), lnPTH (β = −0.133, P = 0.018) and HDL (β = −0.133, P = 0.028). In women, uACR did not affect 25(OH)D3 levels the most, and the parameters were age (β = 0.224, P = 0.004), lnPTH (β = −0.233, P = 0.002), P (β = 0.194, P = 0.013), lnACR (β = −0.184, P = 0.016).
We demonstrated in our cross-sectional study that patients with DN had low vitamin D levels compared to controls. As previously reported, vitamin D analogs or high dose of cholecalciferol are useful as anti-proteinuric agents, and we hypothesized that a conventional low dose of cholecalciferol, 800 IU daily, would also be effective.
A total of 46 patients entered the 6-month follow-up period, 22 in the treated group and 24 in the control group. Baseline patient characteristics are shown in
Treated group | Untreated group | P value | |
|
61.1±10.4 | 60.0±12.2 | 0.67 |
|
22(14) | 24(14) | 0.77 |
|
10±6.1 | 12±7.7 | 0.17 |
|
138±21 | 140±21 | 0.87 |
|
79±10 | 81±7 | 0.31 |
|
6.5±1.3 | 8.2±3.0 | 0.02 |
|
7.1±1.4 | 8.2±1.3 | 0.01 |
|
5.05±1.1 | 4.87±1.2 | 0.60 |
|
2.31±1.3 | 2.17±2.0 | 0.78 |
|
1.08±0.3 | 1.16±0.3 | 0.55 |
|
2.8±0.7 | 2.6±0.8 | 0.43 |
|
2.32±0.1 | 2.25±0.1 | 0.02 |
|
1.29±0.2 | 1.25±0.17 | 0.60 |
|
33.5±15.5 | 29.71±10.5 | 0.35 |
|
87±33.01 | 108±32.93 | 0.04 |
|
14.4 (8.72,18.71) | 13.4(8.06,17.61) | 0.79 |
|
97.4 (62.43,476.70) | 114.4 (65.15,324.57) | 0.92 |
The ACR decreased from 97.39 mg/g (62.43–476.70) to 71.65 mg/g (40.40–469.98) at 2 months (P = 0.01) and 120.36 mg/g (33.89–695.26) at 6 months (P = 0.239,
a: The ACRs in the treated group (n = 22) at 2-, 3-, 4.5-, and 6-month follow-up assessments. Error bars represent 95% confidence intervals. b: The ACRs in the treated and control group before and after follow up. c: The eGFRMDRD in the treated and control group before and after follow up.
Comparison of outcome measures between before and after treatment of longitudinal study is shown in
Treated group (n = 22) | Control group(n = 24) | |||||||
baseline | 6 months | P value | baseline | 6 months | P value | |||
|
7.1±1.4 | 7.2±1.4 | 0.86 | 8.2±1.3 | 8.2±1.5 | 0.80 | ||
|
138±21 | 135±12 | 0.38 | 140±21 | 140±19 | 0.92 | ||
|
79±10 | 76±9 | 0.23 | 81±7 | 78±9 | 0.06 | ||
|
5.05±1.1 | 5.5±1.4 | 0.06 | 4.87±1.2 | 5.4±0.9 | 0.01 | ||
|
2.31±1.3 | 1.79±0.95 | 0.05 | 2.17±2.0 | 2.13±2.6 | 0.92 | ||
|
1.08±0.3 | 1.21±0.24 | 0.02 | 1.16±0.32 | 1.21±0.29 | 0.25 | ||
|
2.82±0.7 | 3.18±0.88 | 0.06 | 2.63±0.77 | 3.07±0.81 | 0.00 | ||
|
2.32±0.1 | 2.35±0.1 | 0.66 | 2.25±0.1 | 2.28±0.9 | 0.17 | ||
|
1.29±0.2 | 1.29±0.2 | 0.95 | 1.26±0.17 | 1.18±0.11 | 0.01 | ||
|
33.5±15.5 | 32.09±11.04 | 0.50 | 30.82±9.40 | 45.32±24.03 | 0.004 |
There was no significant difference in serum 25 (OH)D3 levels at baseline between the treated group and the control group. However, levels in the treated group were significantly higher than those in the untreated group (17.56 (12.23, 23.83) ng/ml vs. 10.52 (7.75, 11.42) ng/ml, P = 0.002) at 6 months (
Serum 25(OH)D3 levels at baseline and at the 6-month follow-up assessment in the treated group and in the control group. Error bars represent 95% confidence intervals.
The results above prompted us to explore the relationship between uACR and serum vitamin D concentrations in the treated group. The patients in treated group with increased serum 25(OH)D3 levels (VD3+,
VD3+ and VD3− reflect the rise and fall of serum 25(OH)D3 levels after treatment with cholecalciferol.▪ACR- reflect the decrease of urinary ACR after treatment with cholecalciferol.
Vitamin D is important in electrolyte regulation. There was no significant difference in serum calcium and phosphate during the entire follow-up period in the treated group. At baseline, the serum calcium concentration was 2.32±0.1 (mmol/l) and the serum phosphate concentration was 1.29±0.2 (mmol/l), while at 6 months, the corresponding concentrations were 2.35±0.1(mmol/l) and 1.29±0.2 (mmol/l), respectively (P = 0.66, 0.95, respectively).
This is the first study to demonstrate that serum vitamin D concentrations are significantly lower in diabetic patients with macroalbuminuria, but not in diabetic patients with microalbuminuria. Furthermore, nutritional vitamin D, cholecalciferol at a conventional dose, may play an important role in terms of an antiproteinuric effect in Chinese patients with T2DM. In addition, observed improvements in proteinuria were associated with a rise in vitamin D concentrations.
Vitamin D is known for its role in the regulation of calcium and phosphate, but recent research has revealed its indispensable role also in the regulation of renal function. Preclinical studies have demonstrated the renoprotective function of vitamin D
Vitamin D analogs, mainly paricalcitol, can effectively decrease proteinuria
In addition, Kim et al. reported that high doses of nutritional vitamin D (40000 units weekly), cholecalciferol, can reduce proteinuria in patients with diabetes
The anti-proteinuric effect of vitamin D in DN is due to its ‘non-classical’ effects, which are unrelated to its role in mineral metabolism, the classical vitamin D effect. The ‘non-classical’ effects are mediated by VDR activation
Unexpectedly, the anti-proteinuric effect had disappeared at the 6-month follow-up assessment in the treated group, although there was a trend for lower ACR levels than at baseline in the treated group, or even at the end of the 6-month period, and also for lower ACR levels in the treated group than in the control group. However, these differences were not significant. In fact, in the VITAL trial, although the reduction in the ACR was sustained during the entire treatment phase both in the 1 µg and 2 µg paricalcitol groups, a peak occurred at the third month, and thereafter the ACR in both groups revealed a marked improvement
The likeliest explanation for this phenomenon may lie with 24-hydroxlayse. On the one hand, vitamin D analogs or nutritional cholecalciferol up-regulate serum vitamin D levels, but on the other hand, 24-hydroxlayse is dramatically activated, thus causing an increasing deactivation of active vitamin D
Diabetic nephropathy is characterized by persistent albuminuria. However, not all the patients with microalbuminuria will progress to diabetic nephropathy
We also found that mean 25(OH)D3 concentrations were significantly lower in females than in males, in our cross-sectional study, which was consistent with previous studies that demonstrated a stronger association between low vitamin D levels and females
Limitations of this study need to be considered. Both in the cross-sectional study and in the longitudinal study, the sample size was small. In addition, the follow-up period was short. As enrollment and the follow-up periods mostly occurred in wintertime, this might affect vitamin D concentrations, and might also influence treatment outcome. Discrepancy in the responsiveness to UVB radiation is clear between individuals
Low vitamin D levels are common in patients with T2DM and albuminuria, particularly those with macro-albuminuria. Conventional doses of cholecalciferol, 800 IU, may have antiproteinuric effects on Chinese patients with DN.