Decreased Endogenous Secretory Advanced Glycation End Product Receptor in Type 1 Diabetic Patients: Its possible association with diabetic vascular complications

A total of 67 Japanese type 1 diabetic patients (22 men and 45 women, age 24.0 ± 4.4 years [means ± SD] with duration of diabetes of 14.5 ± 6.2 years and daily urine C-peptide concentration of 5.6 ± 9.2 μg/day) undergoing periodic follow-up examinations at the Diabetes Clinic of Osaka University Hospital and the Osaka Police Hospital were enrolled in this study. The diagnoses of type 1 diabetes were done by diabetologists. All patients were treated with insulin alone and performed at least three or four daily insulin injections. The daily insulin dose was 0.86 ± 0.25 units/kg. As control subjects, we also enrolled 23 age-matched healthy nondiabetic individuals (10 men and 13 women, age 24.9 ± 1.4 years). None of the subjects had any clinical evidence of infection, connective tissue disease, liver dysfunction, or angiopathy. None of the subjects was taking any oral hypoglycemic drugs or antihypertensive, antiplatelet, or lipid-lowering medications at the time of the study. After a full explanation of the study, written informed consent was obtained from each subject. The study was approved by the Ethical Committee for Human Studies at Osaka University Graduate School of Medicine.

Fasting blood samples were collected, and the laboratory analyses were performed by SRL (Tokyo, Japan) as follows. Serum total cholesterol and HDL cholesterol, serum triglyceride, and HbA_1c (A1C) levels were measured using standard laboratory protocols. High-sensitivity C-reactive protein (hs-CRP) concentration was measured by enzyme-linked immunosorbent assay. The intra-assay coefficient of variation for repeated hs-CRP measurements ranged from 0.80 to 1.72%.

Subjects with diabetes submitted urine samples that had been collected at home over the previous 24 h. Written instructions and a careful explanation regarding the procedure for urine collection were given to each subject. Most of the patients were familiar with the method for collecting urine at home. Nevertheless, a urine sample was discarded when there was any doubt about its collection. The 24-h urine samples collected from each subject were used to determine urinary albumin excretion. According to the amount of their daily urinary albumin excretion, patients were classified into the normoalbuminuria group (urinary albumin excretion <30 mg/day), microalbuminuria group (urinary albumin excretion 30–300 mg/day), and macroalbuminuria group (urinary albumin excretion >300 mg/day). The presence of retinopathy was diagnosed by ophthalmologists based on the findings of funduscopy. Smokers were classified as having a current smoking habit.

To measure the concentration of human circulating esRAGE in serum, we used the B-Bridge esRAGE ELISA Kit (manufactured by Daiichi Fine Chemicals, Takaoka, Japan, and distributed by B-Bridge International). Measurements were performed following the manufacturer’s instructions. The intra-assay coefficient of variation for repeated esRAGE measurements ranged from 3.5 to 6.7%.

To estimate early-stage atherosclerosis, ultrasonographic scanning of the carotid artery was performed using an echotomographic system (Toshiba, Tokyo, Japan) with an electrical liner transducer (midfrequency 8.0 MHz). The detection limit of this echo system using 8.0 MHz was 0.1 mm. Scanning of the extracranial common carotid artery, the carotid bulb, and the internal carotid artery in the neck was performed bilaterally from three different longitudinal projections (i.e., anterior oblique, lateral, and posterior oblique) as well as the transverse projections, as reported in our previous studies (19,20). The intima-media thickness (IMT) defined by Pignoli et al. (21) was measured as follows. At each longitudinal projection, the site of the greatest thickness including a plaque lesion was sought along the arterial walls. Three determinations of IMT were conducted at the site of the thickest point and two adjacent points (located 1 cm upstream and 1 cm downstream from the thickest point). These three determinations were averaged (mean IMT). The greatest value among the six mean IMTs (three from the left and three from right) was used as the representative value for each individual. All ultrasound scans were performed by an experienced sonographer, and an experienced physician performed the determination of IMTs on the photographs. These two individuals were unaware of the subject’s study group and clinical characteristics. Reproducibility of the IMT measurement was examined 1 week later in 30 participants with type 1 diabetes by the same sonographer and the same physician. The mean difference in IMT between these two determinations was 0.04 mm, and the standard deviation was 0.07 mm, demonstrating good reproducibility for repeated measurements.

Data are given as means ± SD. Means or proportions for clinical characteristics were computed for the case and control subjects. Data between the two groups were compared by a two-tailed unpaired Student’s t test, and data among more than two groups were compared by a one-way ANOVA followed by Scheffe’s test. Differences in proportions were tested using the χ² test. Because the hs-CRP distribution was skewed to the left, the median concentrations were computed for these parameters, and the significance of any differences between the patient and control subjects was determined using the Mann-Whitney U test. Single linear univariate correlations (Pearson’s correlation coefficients) and forward and backward stepwise multivariate regression analyses were performed to evaluate the relationship between esRAGE and the following variables: sex, age, duration of diabetes, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), smoking habit, A1C, total cholesterol, triglycerides, HDL cholesterol, and hs-CRP (logarithmically transformed data). Single linear univariate correlations and forward and backward stepwise multivariate regression analyses were performed to evaluate the relationship between meanIMT and the following variables: sex, age, BMI, SBP, DBP, smoking habit, A1C, total cholesterol, triglycerides, HDL cholesterol, hs-CRP (logarithmically transformed data), and esRAGE. For the forward and backward stepwise multivariate regression analyses, the F value for the inclusion and exclusion of variables was set at 2.0. These statistical analyses were performed using Stat-View statistical software (version 5.0 for Windows; Abacus Concepts, Berkeley, CA) and HALBOU statistical software (Gendai Sugaku-sha, Kyoto, Japan) on a personal computer. The threshold of statistical significance was defined as P < 0.05.

Clinical and biochemical characteristics of the study subjects are presented in Table 1. BMI, DBP, A1C, triglyceride levels, and hs-CRP levels were significantly higher in subjects with type 1 diabetes than in nondiabetic subjects (P < 0.05). There was no significant difference between the two groups regarding the other clinical parameters such as age, sex, current smoking habit, SBP, total cholesterol, and HDL cholesterol. Circulating esRAGE levels were significantly lower in subjects with type 1 diabetes than in nondiabetic subjects (0.266 ± 0.089 vs. 0.436 ± 0.121 ng/ml, P < 0.0001) (Fig. 1A). Furthermore, serum esRAGE levels were inversely correlated with A1C levels (r = −0.614, P < 0.0001) (Fig. 1B). Serum esRAGE levels were also inversely correlated with BMI (r = −0.363, P = 0.0005), DBP (r = −0.274, P = 0.0095), triglyceride levels (r = −0.252, P = 0.0175), and the common logarithm of hs-CRP (r = −0.314, P = 0.0024). Furthermore, we performed a stepwise multivariate regression analysis and found that BMI (F = 9.0, P = 0.0035) and A1C (F = 46.1, P < 0.00001) were independent risk factors for a low esRAGE value (Table 2).

Mean IMT was significantly greater in patients with type 1 diabetes than in nondiabetic subjects (0.63 ± 0.11 mm vs. 0.58 ± 0.06 mm, respectively, P = 0.0396). Furthermore, circulating esRAGE levels were inversely correlated with mean IMT (r = −0.325, P = 0.0017) (Fig. 2B). When only the diabetic subjects were analyzed, the correlations between esRAGE and IMT or A1C were weakened. However, there were still statistically significant correlations between esRAGE and IMT (r = −0.289, P = 0.0171) or A1C levels (r = −0.370, P = 0.019). Positive correlations were also observed between mean IMT and sex (r = 0.271, P = 0.0098), BMI (r = 0.256, P = 0.0165), and DBP (r = 0.284, P = 0.0070). To demonstrate that esRAGE is a determinant of mean IMT independent of conventional risk factors, we performed a stepwise multivariate regression analysis and found that esRAGE (F = 4.6, P = 0.035) and sex (F = 4.1, P = 0.047) were variables that interacted independently of mean IMT in all subjects. Taken together, these results show that the esRAGE level inversely correlates with the severity of macroangiopathy.

Circulating esRAGE levels in patients without retinopathy (n = 43), with background retinopathy (n = 20), and with proliferative retinopathy (PDR) (n = 4) were 0.286 ± 0.092, 0.244 ± 0.068, and 0.162 ± 0.067 ng/ml, respectively. There was a significant difference (P = 0.0124) in circulating esRAGE levels between subjects without retinopathy (0.286 ± 0.092 ng/ml) and with retinopathy (background retinopathy + proliferative retinopathy groups, 0.230 ± 0.074 ng/ml) (Fig. 2A). Although the duration of diabetes was significantly longer in the subjects with retinopathy than in those without it (17.3 ± 4.0 vs. 12.9 ± 6.7 years, P = 0.0049), there was no significant difference among the groups in all other parameters.

On the other hand, there was no significant difference in circulating esRAGE levels between patients with microalbuminuria (n = 9, 0.287 ± 0.086 ng/ml) and without it (n = 53, 0.266 ± 0.087 ng/ml). Also, there was no significant difference in all other parameters between the groups. Although there was a weak inverse correlation between circulating esRAGE levels and daily urinary albumin excretion, it did not reach statistical significance (r = −0.242, P = 0.060).

In this study, we found that circulating esRAGE levels were significantly lower in young subjects with type 1 diabetes than in nondiabetic subjects (0.266 ± 0.089 vs. 0.436 ± 0.121 ng/ml, P < 0.0001) (Fig. 1). Furthermore, univariate regression analysis showed a strong inverse correlation between circulating esRAGE and A1C levels (r = −0.614, P < 0.0001). Because circulating esRAGE levels also correlated with BMI, DBP, triglyceride levels, and the common logarithm of hs-CRP, we performed a stepwise multivariate regression analysis and found that A1C (F = 46.1) and BMI (F = 9.0) were independent risk factors for a low esRAGE value. However, whether there is a direct relationship between A1C or BMI and circulating esRAGE levels should be confirmed by further study. It would be necessary to examine the relationship between AGE levels and circulating esRAGE levels before we discuss what factors affect circulating esRAGE levels.

The present study showed that circulating esRAGE levels were inversely correlated with carotid IMT, indicating that esRAGE is inversely correlated with the severity of macroangiopathy. Although carotid IMT is one of the most reliable markers of atherosclerosis, further study using some other indicators of vascular function would be necessary to clarify the relationship between circulating esRAGE levels and macroangiopathy.

The present study also showed that circulating esRAGE levels were significantly lower in subjects with retinopathy than in those without retinopathy (Fig. 2A). In addition, our data suggest that circulating esRAGE levels tend to be inversely correlated with daily urinary albumin excretion. However, another study would be necessary to conclude whether there are some correlations between esRAGE levels and the severity of nephropathy.

It was reported that the C-truncated form of RAGE binds to AGEs and neutralizes their action, leading to vascular dysfunction (11,14,16,17). Furthermore, it was reported that the treatment with C-truncated RAGE stabilizes the progression of atherosclerosis in a mouse model (13,18,22). Taking into account such reports and our present results, it is possible that human circulating esRAGE levels influence the development of diabetic vascular complications. However, further study would be necessary to prove whether diabetic patients with lower circulating esRAGE levels are more susceptible to diabetic vascular diseases.

In summary, circulating esRAGE levels were significantly lower in type 1 diabetic patients than in nondiabetic subjects. The esRAGE levels were inversely correlated with the severity of macroangiopathy and also tended to be inversely correlated with microangiopathy.