Steno Diabetes Center, Gentofte, Denmark
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Abstract |
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Methods. One hundred and sixty patients with type 2 diabetes mellitus and persistent microalbuminuria were followed for an average of 3.8 (SD 0.3) years. 70% of the patients were treated with angiotensin converting enzyme (ACE)-inhibitors. Patients in this subanalysis were divided into two groups according to baseline plasma von Willebrand factor levels below or above the median. The main outcome was cardiovascular disease (cardiovascular mortality, non-fatal stroke, non-fatal myocardial infarction, coronary artery bypass graft and revascularization or amputation of legs), progression to diabetic nephropathy or progression in diabetic retinopathy.
Results. At baseline the two groups were comparable for HbA1c, fasting levels of s-total-cholesterol, s-HDL-cholesterol and s-triglycerides, systolic and diastolic blood pressure, gender, known diabetes duration, smoking habits, previous cardiovascular disease and antihypertensive therapy as well as retinopathy. Odds ratio for cardiovascular disease was 1.11 (95% CI 0.452.73, P=0.82) (multiple logistic regression), odds ratio for progression to nephropathy was 1.08 (0.412.85, P=0.87) and odds ratio for progression in retinopathy was 0.96 (0.462.00, P=0.92), all with plasma von Willebrand factor levels above the median.
Conclusions. Our results do not support the suggestion that the combination of high plasma levels of von Willebrand factor and microalbuminuria is a stronger predictor for cardiovascular disease, progression to diabetic nephropathy or progression in diabetic retinopathy than microalbuminuria alone in patients with type 2 diabetes and persistent microalbuminuria.
Keywords: cardiovascular disease; endothelial dysfunction; microalbuminuria; microvascular complications; type 2 diabetes mellitus; von Willebrand factor
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Introduction |
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Subjects and methods |
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Patients
All 160 participating patients were recruited from the Steno Diabetes Center during 199293. Microalbuminuria was defined as a urinary albumin excretion rate (AER) of 30300 mg per 24 h in four of six 24 h urine samples. Diabetes was defined by 1985 WHO criteria. Exclusion criteria were age older than 65 or younger than 40 years; a stimulated serum C-peptide concentration less than 600 pmol/l 6 min after intravenous injection of 1 mg of glucagon; pancreatic insufficiency or diabetes secondary to pancreatitis; alcohol abuse; non-diabetic kidney disease; malignancy; or life-threatening disease with death probable within 4 years. Informed consent was obtained from all participants. The protocol was in accordance with the Helsinki declaration and was approved by the ethics committee of Copenhagen County.
In the present post-hoc analysis, patients were divided into two groups according to baseline plasma vWF below the median or above the median in the cohort as originally proposed by Stehouwer et al. [4].
Assessments
Endpoint examinations were planned and performed at baseline and after 4 years of follow-up unless otherwise indicated. Blood pressure was measured twice after 20 min rest in the supine position with a Hawksley random-zero sphygmomanometer. Every second year, AER was measured in three consecutive 24 h urine collections. Retinal photographs of two 4550 degree fields (macula-temporal and disc-nasal) were taken in both eyes through dilated pupils. The photographs were graded by two independent ophthalmologists, masked to the original treatment allocation, according to the EURODIAB six-level grading scale [8]. The resting 12-lead electrocardiogram was classified by the Minnesota code [9] by two independent, masked graders. Cause of death was taken from the death certificate. Cardiovascular death was defined as codes 390459 according to the International Classification of Diseases, 9th edn. WHO criteria were used to define acute myocardial infarction and stroke. Procedures were those noted in hospital discharge letters.
Assays
Unless otherwise stated all blood samples were taken at 0800 after an overnight fast. Patients did not take their drugs in the morning of the day of blood sampling. HbA1c was measured by ion-exchange high-performance liquid chromatography (Bio-Rad VARIANT, California, USA) and the non-diabetic reference range in our laboratory was 4.16.4%. Serum total cholesterol and serum HDL-cholesterol concentrations were measured by chromatography. Triglycerides were measured with colorimetry. AER was measured by ELISA [10]. Serum creatinine was measured with routine methods. Plasma vWF was measured by ELISA, as described previously [11].
Endpoints
Cardiovascular disease was as in the original study by Stehouwer et al. [4] a combined endpoint in this case consisting of cardiovascular mortality, non-fatal stroke, non-fatal myocardial infarction, coronary artery bypass graft and revascularization or amputation of legs. Diabetic nephropathy was diagnosed if median AER was greater than 300 mg per 24 h in at least one of the 2-yearly examinations. Progression in diabetic retinopathy was an increase of at least one level in any eye or development of new diabetic retinopathy.
Statistical analysis
Comparison of groups at baseline was by one-way analysis of variance or KruskalWallis test whenever appropriate for numerical variables. 2-test was used to compare categorical variables. Changes in variables during follow-up were compared using Student's t-test or MannWhitney test. For endpoints, odds ratios with 95% confidence intervals were calculated from multiple logistic regression analysis with HbA1c, age, gender, known diabetes duration, smoking habits, original treatment allocation (intensive therapy or standard therapy) and the endpoint of interest at baseline as covariates. Thus, adjustments were made for imbalances between groups at baseline and during follow up in this post-hoc analysis.
Since treatment with angiotensin converting enzyme (ACE)-inhibitors is a potential confounder of plasma vWF levels [12], odds ratios for endpoints were also adjusted for ACE-inhibitor treatment during follow-up in another regression model. Treatment with insulin may also affect circulating levels of vWF [13], therefore a third regression model was adjusted for insulin treatment during follow-up.
To test whether a linear interaction exists between levels of plasma vWF and any of the three endpoints, plasma vWF concentration were entered in the multiple logistic regression model instead of the grouped variable. A non-linear association between plasma vWF levels and endpoints was examined with polynomial regression by entering both plasma vWF levels and squared plasma vWF levels in the model.
P-values less than 0.05 were considered statistically significant.
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Results |
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As seen from Table 2, the two groups with plasma vWF below and above the median, respectively, did not differ significantly in the changes seen in clinical and biochemical variables during follow-up, although weight gain tended to be greater in the group with plasma vWF below the median.
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As shown in Table 3, adjustments for treatment with ACE-inhibitors or insulin during follow-up did not change the odds ratios for endpoints with elevated plasma vWF significantly.
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Discussion |
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In the cross-sectional kidney biopsy study, all microalbuminuric patients with typical diabetic glomerulopathy had diabetic retinopathy and proliferative retinopathy was only present in these patients. However, patients with atypical patterns of renal injury or normal renal structure were equally divided into a group having simplex background retinopathy and a group without diabetic retinopathy [5]. Both typical and atypical renal lesions were associated with worse metabolic control, but furthermore, plasma vWF levels in patients with proliferative retinopathy were significantly higher than in patients without retinopathy [5,14]. In our prospective study we did not see any significant associations with elevated levels of plasma vWF and progression in diabetic retinopathy.
All patients in the intensively treated group and 47% of the patients in the control group in the Steno Type 2 Study were treated with ACE-inhibitors. ACE inhibition has been shown to counteract endothelial activation in hypertensive patients with impaired glucose tolerance [12], and recently treatment with the ACE-inhibitor ramipril has been shown to decrease mortality in high-risk patients, including patients with diabetes [15]. A subanalysis in diabetic patients showed a significant decrease of 16% with 10 mg ramipril daily on a composite endpoint of diabetic complications compared to placebo.
It is noteworthy that, in the original study proposing the term malignant microalbuminuria, only a few of the patients received treatment with ACE-inhibitors [4]. Another epidemiological study which found an association between elevated levels of vWF and cardiovascular mortality in both diabetic and non-diabetic subjects, does not give any information about medical treatment [16]. Finally, a recent study from the same group which found the same association in type 2 diabetic patients with microalbuminuria has excluded patients treated with ACE-inhibitors [17].
It should be noted that there was a perfect match of patients from the intensively treated group and in patients treated with ACE-inhibitors during follow-up between the malignant and benign microalbuminuria group in the present study. Furthermore, adjustment for treatment with ACE-inhibitors during follow-up did not give any significant association between elevated plasma vWF levels and any of the endpoints. However, statistical adjustment may not obviously in all cases sufficiently compensate for biological complexity.
The number of cardiovascular events as defined previously was 29 in the 160 type 2 diabetic patients with persistent microalbuminuria included in the present study giving an odds ratio of 1.11 (95% CI 0.452.73) with elevated plasma vWF. In comparison, 11 events in 28 patients were observed in the original study by Stehouwer et al. [4], which gave a relative risk with elevated levels of plasma vWF of 3.66 (1.311.9). Assuming an equal difference in risk for cardiovascular disease, in the present study the power would be 85% of detecting this difference at a 5% significance level. Because of the small difference in the number of events between the groups with plasma vWF below and above the median, respectively, the actual power is, however, much lower. Although an 11% difference in the risk for a CVD event with elevated plasma vWF could prove clinically important, it should be noted that the 95% confidence intervals are much narrower in the present study than in the study by Stehouwer et al. [4]. We did not have sufficient power to detect an association between elevated levels of vWF and cardiovascular mortality as have been found in other studies [16,17], since only five patients died from cardiovascular causes during follow-up.
It should again be mentioned that the design of our study is a post-hoc analysis and as such is a limitation of our results. The question of selection bias is also important, however inclusion criteria in the Steno type 2 study was simply type 2 diabetes and microalbuminuria, and we therefore do not believe that selection bias is a major problem in this case.
In conclusion, the results of this study with a mean follow-up time of 3.8 years do not support the hypothesis that phenotypic heterogeneity in type 2 diabetic patients with microalbuminuria and, in most cases, concomitant treatment with ACE-inhibitors can be explained by differences in plasma vWF levels. Thus, according to our results, there is no case for benign or malignant microalbuminuria as suggested [6]. However, further large-scale, long-term, prospective studies are required to validate this conclusion.
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Notes |
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References |
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