Simvastatin Reduces Plasma Osteoprotegerin in Type 2 Diabetic Patients With Microalbuminuria

Informed consent was obtained from all participants, and the study received ethics committee approval. Eighteen type 2 diabetic patients were randomly recruited from the outpatient clinic (16). Inclusion criteria were microalbuminuria (overnight urinary albumin excretion 15–200 μg/min), plasma cholesterol ≥5.5 mmol/l, plasma triglyceride ≤4.5 mmol/l, A1C <10%, serum C-peptide >0.49 nmol/l, and blood pressure ≤160/95 mmHg.

The study design has previously been described (16). In brief, in a randomized, double-blind design, patients were allocated to treatment with 10 mg/day simvastatin or the placebo group for 18 weeks. If plasma cholesterol was ≥5.2 mmol/l at 6 weeks, the dose was doubled. Blood samples for OPG, VCAM-1, and ICAM were collected after an overnight fast at baseline and week 18. Sampling for OPG was insufficient for one patient in the placebo group. Sample size was based on previously decided main outcome measurements of renal function (16). Since no interventional studies describing changes in OPG could be identified from the literature, we were unable to perform a valid sample size calculation. We therefore included all patients from that study.

OPG was measured by a sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) using a mouse anti-human OPG as capture antibody and a biotinylated goat anti-human OPG for detection. Recombinant human OPG was used for calibration. Samples were diluted and measured in duplicate (8). Serum ICAM and VCAM-1 were measured by monoclonal antibody–based enzyme-linked immunosorbent assays as described by the manufacturer (catalog nos. BBE1B, BBE3, and DY809, respectively; R&D Systems).

Data are presented as means ± SEM. Between-group differences were analyzed using Student's t test or the Mann-Whitney two-sample test. Changes were also evaluated as the 18 weeks–to–baseline ratio. Correlations were evaluated by Pearson's r.

The treatment groups were similar with respect to age, sex, diabetes duration, BMI, A1C, and serum C-peptide. The average simvastatin dose was 12.5 mg/day. Cholesterol was significantly reduced by simvastatin. A1C remained unchanged.

OPG levels were comparable at baseline (1,660 ± 161 vs. 1,961 ± 131 pg/ml for the placebo vs. simvastatin groups, respectively) and after 18 weeks (1,684 ± 154 vs. 1,816 ± 95 pg/ml), and within-group changes were not statistically significant. However, simvastatin treatment was associated with a significant reduction of baseline–to–18 weeks OPG ratio compared with that in the placebo group (Fig. 1).

No significant differences were observed at baseline in VCAM-1 (755 ± 64 vs. 690 ± 51 ng/ml for the placebo vs. simvastatin groups, respectively) or ICAM (307 ± 83 vs. 336 ± 33 ng/ml). Moreover, the 18 weeks–to–baseline ratios were comparable between groups.

There was no significant correlation between the change in cholesterol and OPG ratio in the simvastatin group or between total cholesterol and OPG, VCAM-1, or ICAM at baseline in the combined group. OPG and A1C tended to correlate at baseline (r² = 0.46, P = 0.06) and week 18 (r² = 0.48, P = 0.07). Plasma insulin (picomoles per liter) correlated significantly with OPG at week 18 (r² = 0.56, P < 0.05).

In this study, low-dose simvastatin treatment for 18 weeks reduced OPG levels in type 2 diabetic patients with microalbuminuria and mild hypercholesterolemia. To our knowledge, this has not previously been demonstrated in humans in vivo.

The cellular mechanism whereby statins affect OPG is unclear. Statins modulate inflammatory mediators on OPG secretion. Thus, simvastatin reduces TNF-α–induced OPG levels in vitro (12) and directly inhibits the nuclear factor-κB system (12,17). Moreover, OPG mRNA and RANKL mRNA were increased in mouse bone-cell cultures incubated with simvastatin for 7–16 days (18). These results seem contrary to ours, since we found decreased OPG levels. However, the different study design (in vitro vs. in vivo) and treatment duration (7 and 16 days vs. 18 weeks) may partly explain the differences. Moreover, since OPG is involved in calcification inhibition, at least in mice (10), our findings may reflect a simvastatin-mediated calcification reduction, which, in turn, might downregulate OPG. In type 2 diabetes, microalbuminuria is not known to be related to altered bone mineral metabolism.

Contrary to previous reports (19), we found no change in VCAM-1 or ICAM. These adhesion molecules are induced by inflammatory cytokines (i.e., TNF-α and interleukin-1), oxidative stress, and oxidized LDL (20). In theory, the anti-inflammatory and LDL-lowering effects of simvastatin should, therefore, exert an inhibitory effect on adhesion molecule expression (17,19). To our knowledge, this has never been shown in human in vivo studies. Mulder et al. (21) recently reported that switching to more aggressive lipid lowering in patients already on statin treatment may not have further effects on adhesion molecule expression. We included statin-näive patients in the present study. Although the inclusion criteria do not adhere to today's standards of clinical care, we think that the interpretations of the results are not hampered in a major way. Our inability to show changes in adhesion molecules may be due to the relatively low statin dose, the study duration, or the number of patients included.

In summary, 18 weeks of low-dose simvastatin treatment reduced circulating OPG levels in type 2 diabetic patients with microalbuminuria but had no effect on VCAM-1 or ICAM. The reduction of OPG was independent of cholesterol and suggests a pleiotropic effect of simvastatin per se. The OPG-lowering effect of simvastatin may signal diminished vascular calcification.

This study was supported by Novo Nordisk Research Foundation, the Danish Medical Research Council, and Merck Sharp & Dohme.

We thank L. Larsen, A. Mengel, and M. Møller for technical assistance.