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Vascular complications are the main causes of morbidity and mortality in patients with diabetes. It has been very well established that microalbuminuria is defined as an abnormally elevated AER below the level of clinical albuminuria; this represents a urinary albumin excretion rate of 20–200 μg/min, equal to 30–300 mg/24 h (1). In patients with IDDM, microalbuminuria predicts the development not only of diabetic nephropathy but also of macrovascular disease (2, 3).

There are several indications that endothelial damage could play a key role in the development of both micro- and macroangiopathy (4). The endothelium is an important locus of control of vascular functions. It actively regulates vascular tone and permeability, the balance between coagulation and fibrinolysis, the composition of the subendothelial matrix, and the mitogenesis of vascular smooth muscle and renal mesangial cells (5–7). It has been shown that there is a close association between microalbuminuria and endothelial dysfunction (2, 3, 8).

Thus, in patients with microalbuminuria, the vascular endothelium tends to increase vascular resistance (9, 10), fails to restrict the passage of macromolecules (2, 3, 8), and loses its anticoagulant and profibrinolytic properties (11, 12). In adult patients with diabetic nephropathy, a significant increase in plasma concentration of markers of endothelial injury and dysfunction, such as vWF, a glycoprotein involved in primary hemostasis and secreted mainly by endothelial cells and megakaryocytes, has been shown (11, 13).

Some authors have suggested that measuring vWF can have a role in the prediction of risk for development of nephropathy (14, 15), whereas others did not confirm this hypothesis (16, 17). vWF is synthesized as a large 360-kD precursor, named pro-vWF. After many changes in the endoplasmic reticulum and the Golgi apparatus, this precursor undergoes cleavage into mature vWF and a large 97-kD propeptide. Stimulation of exocytosis causes the release of equimolar amounts of vWF and propeptide. It has been suggested (18) that concomitant determinations of plasma vWF and its propeptide are useful tools to assess endothelial activation in vivo, but there is no study in which the levels of propeptide have been evaluated in diabetic children.

The aims of the present study were to evaluate whether endothelial dysfunction precedes the occurrence of microalbuminuria and whether it can help to identify those patients who are at risk to develop microalbuminuria.

METHODS

Subjects.

One hundred eleven IDDM patients (52 girls and 59 boys) were recruited prospectively from the Department of Pediatrics, University of Chieti. During the study nine of 111 children showed intermittent microalbuminuria (defined as AER > 20 μg/min in one of three overnight urine collections in the last 6 mo and with a persistence of normoalbuminuria in the next years), and for this reason we excluded them from the study. Therefore, the evaluation was performed on the remaining 102 children. Their age ranged from 8.1 to 12.9 y with a duration of diabetes of 4.0 to 9.9 y. Inclusion criteria for study participation were absence of other metabolic diseases and normal liver and renal function tests, absence of any medication (including nonsteroidal antiinflammatory drugs or angiotensin-converting enzyme inhibitors), use of no medication other than insulin, duration of disease longer than 5 y, and normal blood pressure. The exclusion criteria were diabetic retinopathy on fluorescein angiography, presence of microalbuminuria and nephropathy, and electrophysiologic abnormalities of autonomic or peripheral neuropathy. Patients were divided into two groups according to the presence or absence of microalbuminuria at the end of follow-up. No patient smoked at the beginning of the study and during the whole follow-up. Control group consisted of 80 age- and sex-matched healthy volunteers. They did not receive any drug before blood collection. Clinical and laboratory data were obtained at baseline (baseline evaluation) and during 8 y of follow-up (follow-up evaluation). Clinical (diabetes duration, blood pressure, insulin dose) as well as laboratory data (glucose, fibrinogen, vWF, vWF propeptide, HbA1c, serum creatinine concentration, AER) were evaluated. At the beginning of the study there were no differences between the two groups of patients in age, sex, duration of diabetes, and serum creatinine (Table 1).

Table 1 Pertinent data of the two groups of diabetic patients and of control subjects
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During the study, patients did not undergo antihypertensive therapy and did not show clinical or biochemical evidence of renal disease. At the beginning of the study all patients showed an AER < 20 μg/min in three overnight urine collections obtained over 6 mo. We defined persistent microalbuminuria as an AER > 20 μg/min in two of three consecutive overnight urine collections. All patients had normal blood pressure values. The informed consent of study has been obtained from the parents of all patients studied; consent was obtained also in the control group. The study was approved by the Ethical Committee of the Faculty of Medicine, University of Chieti, Italy.

Methods.

AER was measured by an ELISA technique (19), and the annual level was determined as the mean of AER in three overnight urine collections taken at home during normal physical activity. The interassay and intraassay variations of AER measurement were 8.3 and 2.1%, respectively. Plasma fibrinogen and vWF were measured centrally on citrated plasma prepared from fresh blood obtained by venepuncture. Plasma samples were stored at −20°C at all times until assay. Fibrinogen was assayed by a clotting assay based on the prothrombin time, on an Instrumentation Laboratories Series 300 Autoanalyzer, and vWF antigen was assayed by ELISA, as previously described (20); a pooled plasma standard, calibrated against the U.K. (NIBSAC) standard, was used. vWF propeptide was measured by a method described in detail previously (21). Briefly, 96-well plates were coated with the MAb BR5 and 3H4, 0.75 μg/mL each in carbonate buffer (NaHCO3, 50 mM, pH 9.6). After blocking of the remaining protein binding sites with 5% (wt/vol) dry skim milk, the samples and standards diluted in blocking solution were added and incubated for 2 h at 37°C. The bound antigen was detected with a third antipropeptide MAb (8H10) conjugated to alkaline phosphatase diluted 1:800 (approximately 0.25 μg/mL). Alkaline phosphatase activity was detected using p-nitrophenyl phosphate as a substrate and measuring the OD at 405 nm. Using purified plasma propeptide as a standard, we determined the propeptide concentration in normal pooled plasma at 720 ± 43 ng/mL (mean ± SEM, n = 5). For routine assays, a standard curve was constructed with normal pooled plasma. Each sample was tested at four dilutions of 1:320–1:640, which corresponds to a sensitivity of <2.5 ng/mL (<25 pmol/L, assuming a molecular weight of 97 kD).

HbA1c was measured by HPLC (DIAMAT; Bio-Rad, Richmond, CA, U.S.A.). The normal range was 4.1 ± 1.4%. Blood pressure was measured by a standard clinical sphygmomanometer on the right arm after a 10-min supine rest. Serum creatinine was measured by Jaffe's method to exclude pseudocreatinine. Glucose was determined instantaneously by the glucose oxidase–based method (YSI, Yellow Springs, OH, U.S.A.) in arterial whole blood.

Statistical analysis.

Data are presented as mean ± SD or medians with ranges. χ2 tests, two-tailed t test, or the Mann-Whitney nonparametric test was used to assess the degree of statistical significance of differences. In particular, the two groups were compared with regard to laboratory characteristics and changes therein at each examination using standard parametric and nonparametric testing as indicated by Bonferroni's test.

Pearson's coefficients were calculated for correlation analyses. Probability values <5% were considered statistically significant. All analyses were run on a personal computer with the statistics package SPSS for Windows version 5.0.

RESULTS

Baseline evaluation.

At the beginning of the study, patients with IDDM showed normal AER (<20 μg/min). Fibrinogen, vWF, and vWF propeptide values were similar to controls, and there were no significant differences for all variables studied.

Follow-up evaluation.

During 8 y and at the end of follow-up, 19 of the 102 patients (18.6%) developed persistent microalbuminuria. An increase of plasma vWF and vWF propeptide levels was observed in patients who later developed microalbuminuria (group B) compared with those who remained normoalbuminuric (group A; Figs. 1 and 2). This increase started 3 y and become statistically significant (p < 0.01) 2 y before the onset of persistent microalbuminuria, persisting until the end of the study. In detail, at the end of the study, in group A we observed six of 83 children who had vWF and its propeptide higher than 170 U/L and 175%, respectively; in group B two of 19 patients who had vWF and its propeptide lower than 170 U/L and 175%, respectively. The positive predictive value was 89.5%, and the negative predictive value was 88.0%.

Figure 1
figure 1

Plasma vWF levels in group A (▪) and group B (•) patients. Year 0 is the year of the beginning of persistent microalbuminuria.

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Figure 2
figure 2

Plasma vFW propeptide levels in group A (▪) and group B (•) patients. Year 0 is the year of the beginning of persistent microalbuminuria.

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The increase of AER was more evident during the pubertal development of these patients. Moreover, nine of the 19 patients showed the development of background and preproliferative retinopathy (four and five patients, respectively) evaluated by fluorescence angiography.

During the entire period of the study, plasma values of fibrinogen persisted in the normal range both in microalbuminuric and in normoalbuminuric patients (Fig. 3).

Figure 3
figure 3

Plasma fibrinogen levels in group A (▪) and group B (•) patients. Year 0 is the year of the beginning of persistent microalbuminuria.

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In both groups a significant increase of HbA1c values was observed at the end of the study (Fig. 4). Values of AER during the study are shown in Figure 5.

Figure 4
figure 4

HbA1c percentages in group A (▪) and group B (•) patients. Year 0 is the year of the beginning of persistent microalbuminuria.

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Figure 5
figure 5

AER values in group A (▪) and group B (•) patients. Year 0 is the year of the beginning of persistent microalbuminuria.

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To evaluate the relationship between age, diabetes duration, sex, HbA1c, blood pressure, and onset of microalbuminuria or retinopathy and fibrinogen, vWF, and its propeptide levels, we performed a multivariate analysis (ANOVA): no significant relationship was found.

DISCUSSION

Injury to the vascular endothelium appears to be central to the pathogenesis of atherosclerosis and may be an initiating event. Endothelial dysfunction is also a feature of diabetic nephropathy, even during the subclinical phase.

It has recently been shown that adult IDDM patients with albuminuria have elevated vWF and fibrinogen plasma concentrations (15, 18). In the present follow-up study, we found an association between the increases of AER, vWF, and its propeptide before the development of persistent microalbuminuria by as much as 2 y.

vWF and vWF propeptide are released in equimolar amounts; parallel determination of plasma vWF and its propeptide can help to differentiate between increased endothelial secretion and alterations in vWF catabolism (18). We measured both vWF and vWF propeptide, demonstrating that endothelial dysfunction is present at least 2 y before the development of persistent microalbuminuria. Some authors (18, 21) suggested that in conjunction with vWF, vWF propeptide may improve the identification of endothelial activation; our data suggest that vWF propeptide concentrations do not increase earlier than vWF values before the onset of microalbuminuria, but its increase is simultaneous to the increase of vWF concentrations. Therefore, the parallel determination of vWF and vWF propeptide does not add any useful information for the early prediction of the development of microalbuminuria.

Stehouwer et al.(14) reported that the increase in plasma vWF precedes and may therefore help to predict the development of microalbuminuria in IDDM. More recently, Greaves et al.(15) found a positive relationship among fibrinogen, vWF, and AER in those with retinopathy but not in those without retinopathy. These findings are consistent with the hypothesis that microalbuminuria and increased vWF and vWF propeptide are related to endothelial cell perturbation in response to mildly raised blood pressure in subjects with retinopathy. Fibrinogen may also contribute to microvascular disease, and its relationship to lipid vascular risk factors suggests a possible pathogenetic role in vascular complications of diabetes.

Endothelial dysfunction in microalbuminuric IDDM patients appears to be generalized in that it affects many aspects of endothelial function (2, 8, 9, 11, 22). We suggest that endothelial dysfunction in IDDM develops gradually, with sustained increases in plasma vWF, its propeptide, and AER as relatively advanced features.

The type of endothelial dysfunction reflected by increased vWF and fibrinogen levels is closely related to microalbuminuria not only in IDDM (13) but also in non–insulin-dependent diabetes mellitus (22, 23) and essential hypertension (24), although all our patients remained normotensive until the end of follow-up.

It is not clear, however, whether the prognostic value of vWF and vWF propeptide is related to their specific functions, i.e. enhancement of platelet adhesion and factor VIII availability, or whether they are simply markers of endothelial injury and dysfunction. Nevertheless, vWF, vWF propeptide, and AER deserve consideration as clinically useful markers of vascular status.

It has long been recognized that plasma fibrinogen concentrations are elevated in diabetes (4) but a relationship with nephropathy has not always been apparent (11). Fibrinogen could contribute to the development of nephropathy in a variety of ways: it is the substrate for thrombin in blood coagulation, and animal studies indicate that the fibrinogen concentration at the initiation of coagulation influences the amount of fibrin deposited (14). Our study demonstrates that fibrinogen does not increase in patients who develop microalbuminuria.

CONCLUSION

In conclusion, the present data show that in adolescents and young adults with onset of diabetes during childhood, endothelial dysfunction, evaluated as vWF and vWF propeptide, precedes the development of microalbuminuria and may help to identify patients with onset of diabetes during childhood at risk to develop diabetic nephropathy later in life.