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Department of Medicine, Division of Pediatrics, University of Chieti, 66100 Chieti, Italy; and Department of Pediatrics, Oregon Health Sciences University (A.S.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Francesco Chiarelli, M.D., Clinica Pediatrica, Ospedale Policlinico, Via dei Vestini 5, 66100 Chieti, Italy. E-mail: chiarelli{at}unich.it
Abstract
This study was designed to evaluate whether vascular endothelial growth factor serum concentrations may identify adolescents with onset of type 1 diabetes during childhood at greater risk to develop persistent microalbuminuria and incipient diabetic nephropathy. In January 1989, vascular endothelial growth factor serum levels were measured in 101 normoalbuminuric diabetic children and adolescents (aged 7–14.9 yr; onset of diabetes before age 18 yr; duration of diabetes >7 yr). Participants were clinically examined at baseline and annually thereafter. Vascular endothelial growth factor serum concentrations were measured every year during the 8-yr follow-up period. Over 8 yr, 11 of 101 patients (10.9%) developed persistent microalbuminuria; no patient developed overt nephropathy. The risk of developing microalbuminuria was higher in children with increased vascular endothelial growth factor serum levels (using 160 pg/ml as the arbitrary cut-off point; group 1) compared with those with normal vascular endothelial growth factor serum levels at the beginning of the study (group 2; 19.2 vs. 2.0%; P < 0.01; sensitivity, 90.9%; specificity, 53.3%). The odds ratio for the occurrence of microalbuminuria after adjustment for confounding variables (albumin excretion rate, sex, hemoglobin A1c, mean blood pressure, cholesterol, and triglycerides) in type 1 diabetic adolescents with elevated vascular endothelial growth factor serum levels was 4.1 (95% confidence interval, 2.0–10.9).
These results suggest that vascular endothelial growth factor serum concentrations may be one of the predictors and risk factors for microalbuminuria and incipient diabetic nephropathy in adolescents and young adults with onset of diabetes during childhood. Persistently increased vascular endothelial growth factor serum levels may help to identify normotensive, normoalbuminuric patients with type 1 diabetes who are predisposed to develop persistent microalbuminuria later in life.
RENAL DISEASE remains the major cause of morbidity and mortality among patients with type 1 diabetes (1). The cumulative risk for developing diabetic nephropathy is about 30–50% after 40 yr of disease, but those who develop type 1 diabetes during childhood seem to have considerably higher risk (2). Even if diabetic nephropathy is rare in children and adolescents (3, 4), the early stages of this complication can be detected by the presence of microalbuminuria, defined as a urinary albumin excretion rate (AER) of 20–200 µg/min in two of three overnight collections obtained in 6 months (5). Microalbuminuria is the first clinically identifiable sign of risk of developing diabetic nephropathy and other vascular complications, and in particular, it is predictive of the occurrence of overt proteinuria and cardiovascular complications, particularly in IDDM (6).
The identification of high risk patients before the onset of microalbuminuria would have great importance in pinpointing subjects with diabetes who might benefit from a more precocious and aggressive medical treatment aimed at arresting the progression to the later stages of the disease.
Mean glycated hemoglobin (HbA1c) is the dominant predictor of nephropathy, as demonstrated by cross-sectional and longitudinal studies (7, 8). Yet despite maintenance of good metabolic control, 10–15% of patients develop diabetic nephropathy after 20 yr of diabetes duration (8); this evidence suggests that additional factors may contribute to determine the risk of developing diabetic nephropathy, but, unfortunately, provides no explanation for the mechanisms responsible (9).
The structural changes occurring in early diabetic nephropathy, characterized by renal hypertrophy and increased microvascular permeability, suggest a causal role for impairment of the growth factor network (10). A large amount of evidence supports the hypothesis that a panel of growth factors, in particular the GH/IGF-I axis and TGFß isoforms, are involved in the development of diabetic nephropathy through a complex intrarenal system (10). Together with them vascular endothelial growth factor (VEGF) is one of the most likely candidates for the development of both retinopathy and nephropathy, even if evidence for the latter is less extensive to date (11, 12). VEGF is a cytokine with potent vascular permeability and angiogenic effects and has been implicated in the pathogenesis of vascular-related diseases, such as growth of tumors, atherosclerosis of coronary arteries in ischemic heart disease, Kawasaki disease, neovascularization in synovial tissue in rheumatoid arthritis, and diabetic microangiopathy (13, 14).
VEGF expression and binding have been detected in the kidney of rats (12) and humans, in particular in the epithelial cells of the glomerulus, podocyte, and collecting ducts (15). The same stimuli have been shown to increase the expression of VEGF as well as of the other growth factors implicated in the development of diabetic nephropathy: hypoxia, vasopressor hormones such as vasopressin and angiotensin II, advanced glycation end products (AGEs), mechanical strain, cytokines such as TGFß, and growth factors such as fibroblast growth factor and platelet-derived growth factor (10). Furthermore, TGFß and IGF-I have been reported to enhance VEGF production in fibroblastoid and epithelial cells and in retinal cells, respectively (10). These links between VEGF and other growth factors whose roles in the pathogenesis of diabetic nephropathy are well established suggest that VEGF may act in concert with them as a causal factor and eventually may represent a marker for the capacity of an individual to produce a potentially detrimental molecule in the development of diabetic complications.
Increased serum concentrations of immunoreactive VEGF have been described in adults with type 2 diabetes and diabetic nephropathy (16). Data from our group have shown that serum VEGF concentrations are increased in prepubertal and pubertal children with type 1 diabetes, and that there is a direct correlation between serum levels of VEGF and the severity of complications (17).
To the best of our knowledge, no long-term study has been performed in children and adolescents with type 1 diabetes mellitus. In the present study plasma VEGF levels were evaluated in a large group of adolescents with onset of diabetes during childhood to determine whether increasing VEGF levels may predict the occurrence of persistent microalbuminuria.
Subjects and Methods
Patients
The study started in January 1989. The patients were selected from those regularly attending the Department of Medicine and the Department of Pediatrics, University of Chieti (Chieti, Italy). To be eligible for participation, the patients had to fulfill the following inclusion criteria: onset of diabetes before the age of 18 yr, duration of disease longer than 7 yr, insulin treatment from the time of diagnosis, and normal blood pressure.
One hundred and one patients (52 females and 49 males, aged 7–14.9 yr)
satisfied the inclusion criteria and agreed to participate. They were
arbitrarily divided into 2 groups on the basis of VEGF serum
concentrations: group 1 had levels above and group 2 had levels below
160 pg/ml. The sensitivity positive, the predicting value for the
occurrence rate of microalbuminuria, and the specificity negative, the
predicting value for the occurrence rate of microalbuminuria, were
calculated. One hundred and two healthy subjects, matched for age and
sex, were also studied as control groups. The two groups of patients
with type 1 diabetes were comparable for age, duration of diabetes,
body mass index and biochemical parameters,
HbA1c, AER value, and systolic and diastolic
blood pressures. The baseline clinical characteristics of the 2 groups
of type 1 diabetic patients who completed the study are summarized in
Table 1
.
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Patients and their families gave informed consent to participate, and the study was approved by the ethics committee of University of Chieti.
Quantification of VEGF concentrations in serum
VEGF serum levels were measured in all samples in duplicate by a sensitive and highly specific colorimetric ELISA with slight modifications of a chemiluminescence enzyme immunoassay method previously described (18). The recombinant human VEGF121 was purified from the culture medium of the transformed yeast (19). The antihuman VEGF polyclonal antibody was prepared from rabbit serum immunized with the recombinant human VEGF121-glutathione-S-transferase fusion protein (20). The specificity of this anti-VEGF polyclonal antibody was well demonstrated by other investigators (18, 19, 20). As the cross-reactivity between this assay and VEGF165 (which is the most abundant VEGF isoform in the blood) is 92%, we can assess that plasma levels of VEGF121 reliably represent concentrations in serum. The minimal detectable VEGF concentration was 8.0 pg/ml. There were 16 values of VEGF under the detection limit; undetectable values were assigned the detection limit. Intra- and interassay coefficients of variation were 5.1% and 6.2%, respectively. To assess the consistency of VEGF determination over the 9-yr follow-up, we measured serum VEGF concentrations in the same samples after 9 yr, and we achieved similar results (interassay variation, 7.4%).
Other analyses
A number of parameters were determined in all patients at baseline and every 6 months: HbA1c, plasma electrolytes, cholesterol, triglycerides, serum creatinine, AER from three 24-h urine collections, body weight, and arterial blood pressure.
HbA1c was measured throughout the 8-yr study by HPLC (Bio-Rad Laboratories, Inc., Richmond, CA); the mean of at least four HbA1c determinations in 1 yr was considered the index for glycemic control. The mean ± SD HbA1c value in the control population was 4.8 ± 0.1%. Our HbA1c method has been compared with an international reference laboratory at Steno Memorial Hospital in Copenhagen; the correlation between the two laboratories was significant (y = 0.9703x + 0.387; r2 = 0.9709; P < 0.001), as previously described (21). The creatinine concentration was measured by an autoanalyzer (Astra 8, Beckman Instruments, Inc./Hybritech, Palo Alto, CA) (22).
AER was measured by RIA (Pharmacia, Uppsala, Sweden) as described previously (13). Persistent microalbuminuria was defined as an AER more than 20 µg/min in at least two of three overnight urine collections in 6 months. Intermittent microalbuminuria was defined as an AER not constantly more than 20 µg/min in at least three subsequent determinations in 6 months. All subjects were weighed wearing indoor clothing without shoes, and height was recorded as well. Blood pressure was measured according to the recommendations of the Task Force on Blood Pressure Control in Children (23, 24) and the American Heart Association (25). Blood was collected in the morning after an overnight fast and before the usual morning insulin injection.
Patients were advised to follow the same dietary intakes advised for the general diabetic population. A normocaloric diet was prescribed, with carbohydrate intake ranging from 54–60%, protein intake from 12–14%, salt intake less than 120 mEq/liter·73 m-2/d, and cholesterol intake less than 100 mg/1000 calorie. Diet was adjusted on the basis of dietary habits of the families and children; all patients and their families received dietary education to improve dietary compliance and children’s diet, according to the Italian government’s recommended dietary allowances.
Statistical analysis
Values are given as the mean ± SD or as the
median with the range. Comparison between the values observed during
the follow-up period in groups 1 and 2 used a two-factor ANOVA with
VEGF serum level category and time (baseline and follow-up period) as
the factors when a significant interaction was shown by ANOVA. The
2 tests, two-tailed t test, or
Mann-Whitney nonparametric test was used to assess the degree of
statistical significance of differences.
As the descriptive measure of association between risk factors and each of the outcomes, odds ratios (ORs) have been given together with 95% confidence intervals. These ORs were based on the cumulative incidence rates of microalbuminuria and macroalbuminuria during the 8-yr follow-up interval (the average in the two pooled groups). Multiple logistic regression analysis was used to study the relationship between the occurrence of microalbuminuria (dependent variable) and the baseline rate of serum VEGF concentrations (independent variable) while simultaneously controlling for sex and baseline AER and the mean values of plasma creatinine, HbA1c, and systolic and diastolic blood pressure levels every 6 months during the follow-up period. These independent variables were chosen because of their univariate associations with the occurrence rate of microalbuminuria. The occurrence rate of persistent microalbuminuria was treated as a dichotomy (AER >20 µg/min vs. AER <20 µg/min) and as a continuous numerical value [cumulative absolute change in AER (micrograms per min) from baseline]. Statistical analyses were carried out using 6.1 Base System software (SPSS, Inc., Chicago, IL).
Results
VEGF serum concentrations
The median value of VEGF serum levels in the type 1 diabetic
patients who took part in the study (101 subjects) was 160 pg/ml.
Therefore, the cut-off value of 160 pg/ml was used as a reasonable
number to have the most acceptable number of false negative and false
positive results among subjects with diabetes. In healthy control
subjects, the mean ± SD serum VEGF concentration was
117.4 ± 16.3 pg/ml. Of the patients with diabetes at the
beginning of the study, 52 (27 females and 25 males) had values higher
than and 49 (25 females and 24 males) had values lower than the median
level and were assigned to groups 1 and 2, respectively. No significant
changes in serum VEGF levels were observed between baseline, 2-yr,
4-yr, 6-yr, and 8-yr measurements in each group (mean ±
SD, 177.4 ± 26.9, 174.6 ± 23.9, 180.3 ±
26.8, 184.3 ± 21.2, and 186.1 ± 22.7 pg/ml in group 1;
140.1 ± 19.7, 143.4 ± 20.3, 145.6 ± 19.1, 142.1
± 23.4, and 146.2 ± 21 pg/ml in group 2; Fig. 1).
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During the entire follow-up period, group 1 patients had
significantly higher mean HbA1c values than group
2 patients (9.4 ± 1.9 vs. 7.9 ± 2.0;
P < 0.01; Fig. 2).
Systolic and diastolic blood pressure levels were slightly, but
significantly, higher in group 1 than in group 2, although they were
within the normal range (124 ± 12 vs. 118 ± 9 mm
Hg; P < 0.01; Fig. 2). No differences were observed
with regard to other clinical or biochemical parameters.
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). No significant differences were
observed with regard to the rate of development of intermittent
microalbuminuria (Table 2). The risk of developing persistent
microalbuminuria was higher in children with serum VEGF levels higher
than 160 pg/ml (19.2% vs. 2.0%; sensitivity, 90.9%;
specificity, 53.3%). The specificity was better (85.1%) if the
cut-off was fixed at a VEGF concentration of 180 pg/ml, but with this
cut-off value the sensitivity was much lower (76.2%; Table 3). Therefore, the VEGF cut-off value of
160 pg/ml has the most acceptable number of false negative and false
positive results among adolescents and young adults with diabetes
(Table 3). The OR for the occurrence of microalbuminuria after
adjustment for the mentioned confounding variables, such as AER, sex,
HbA1c, mean blood pressure, cholesterol, and
triglycerides, in type 1 diabetic patients with VEGF values higher than
160 pg/ml was 4.1 (95% confidence interval, 2.0–10.9). Similar
results were observed using the cumulative absolute increase in AER
above baseline as the continuous numerical variable.
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The present study shows that patients with increased VEGF serum concentrations have an increased risk to develop persistent microalbuminuria. These results are in agreement and extend previous findings from our group showing that serum levels of VEGF are increased in preschool, prepubertal, and particularly pubertal patients with diabetes compared with controls, and that this increase is related to the severity of nephropathy and retinopathy in adolescents and young adults with onset of diabetes during childhood (17). These reports give relevance to the hypothesis that VEGF could play a pivotal role in the development and progression of vascular complications in diabetes.
In addition to the well established role of this cytokine in both nonproliferative and proliferative retinopathy (10), a large body of evidence in vitro and in animal models suggests that this pathological effector is also induced in other tissues, namely the kidney, by the same or different stimuli. A very recent study indicates an early and persistent increase in renal VEGF gene expression in association with experimental diabetes as well as an early and transient increase in renal VEGF receptors in streptozotocin-induced diabetic rats (12). Because the main biological effect of this growth factor consists in promoting angiogenesis and permeability (14), its overexpression in the kidney might represent an excessive compensatory mechanism in response to renal injury, leading to increased albumin permeability and the occurrence of albuminuria.
It is likely that VEGF together with other growth factors could act as a distal effector of hyperglycemia-accelerated pathways and metabolic perturbations occurring in diabetes. These include increased polyol pathway activity and associated changes in intracellular redox state (13), increased diacylglycerol synthesis with consequent activation of specific PKC isoforms, increased nonenzymatic glycation of both intracellular and extracellular proteins, elevated angiotensin II, hypoxia, increased formation of reactive oxygen species, and mechanical stretch (10). The up-regulation of VEGF expression triggered by the aforementioned stimuli in the kidney provides an explanation and suggests a plausible source for the increased VEGF levels detected in the serum of our patients.
VEGF serum levels are likely to be continuous; the selection of VEGF cut-off of 160 pg/ml is arbitrary and was made in response to the analyses to achieve the most acceptable sensitivity and specificity.
We also found that glycemic control was poorer in patients who more frequently developed persistent microalbuminuria later in life (group 1); in fact, the mean HbA1c values during the entire follow-up were significantly higher in group 1 than in group 2. Chronic hyperglycemia is able to increase the formation and accumulation of AGE in the kidneys of diabetic rats and in the serum of children and adolescents with type 1 diabetes (26); this overexpression of AGE, in turn, may initiate a positive feedback loop for increasing VEGF levels, as improvement of long-term glycemic control is able to reduce VEGF levels in preschool, prepubertal, and pubertal patients. In this perspective, glycemic control may be considered the major determinant of VEGF overexpression in diabetes.
On the other hand, in our study, despite an increase in HbA1c as well as in blood pressure during the follow-up period, no significant changes in serum VEGF levels were observed between baseline, 2-yr, 4-yr, 6-yr, and 8-yr measurements. Therefore, it is unlikely that increased HbA1c or blood pressure in group 1 was able to affect the trend of serum VEGF concentrations, at least for the duration of the observed 8-yr period of follow-up. In accordance with this observation, recent data support the hypothesis of a genetic basis in the regulation of growth factor expression: thanks to the study of TGFß polymorphism, it was demonstrated that the TGFß circulating concentration is predominantly under genetic control, and very recent data show gene polymorphism of VEGF to be correlated with VEGF protein production (27, 28).These findings might relieve environmental factors of the responsibility of growth factor up-regulation, leaving them the role of enhancers, accelerating a process that is already genetically determined.
The persistently elevated VEGF concentrations detected in our study, several years before the onset of persistent microalbuminuria, suggest that expression of VEGF may be at least in part genetically determined and, as such, a valid marker for future development of complications; therefore, further studies are needed on VEGF gene polymorphism in patients with diabetes.
In patients with diabetes, microalbuminuria is a predictor of widespread severe microangiopathy and macroangiopathy. Patients with microalbuminuria show generalized dysfunction of the vascular endothelium, which may underlie the propensity of microalbuminuric patients to develop severe extrarenal vascular disease. Endothelial dysfunction has been shown to be important in the development of microalbuminuria (29, 30). Thus, in cross-sectional studies in patients with type 1 diabetes mellitus and microalbuminuria, the vascular endothelium tends to increase, rather than decrease, vascular resistance (31); fails to restrict the passage of macromolecules (1); and loses its anticoagulant and profibrinolitic properties (30, 32). In addition, there is an increase in the plasma concentrations of markers of endothelial injury and dysfunction, such as von Willebrand factor, a glycoprotein involved in primary hemostasis and secreted mainly by endothelial cells (30, 33); more importantly, endothelial dysfunction, as estimated by plasma von Willebrand factor concentration, precedes the development of microalbuminuria in insulin-dependent diabetic patients by as much as 3 yr (34).
Because microalbuminuria reflects a more generalized vascular process that affects the glomeruli, the retina, and the intima of large vessels simultaneously (1, 35), it is possible to hypothesize that VEGF may represent a reliable and early marker of this generalized vascular dysfunction and may contribute to endothelial damage in young patients with diabetes.
In conclusion, our data suggest, on the one hand, that longitudinal monitoring of a panel of plasma markers such as that used in the current study may better define their relevance in progressive kidney disease and provide greater insight into the mechanisms underlying these processes. On the other hand, a noninvasive estimation of VEGF concentrations may be a reliable method of predicting renal involvement. As microalbuminuria occurs relatively late in the disease process, evaluation of serum concentrations of VEGF may become a useful prognostic test in the earliest stages of the disease and could help to identify children and adolescents at higher risk of developing nephropathy later in life, who might benefit from a earlier and more aggressive medical intervention.
Acknowledgments
We acknowledge the editorial assistance of Mrs. Antonella Bascelli. We also thank Doriana Dalimonte D’Attilio from the Juvenile Diabetes Association (Chieti, Italy) for her invaluable help with the care of children with diabetes.
Footnotes
This work was supported by grants from the Italian Ministry for University and the local Juvenile Diabetes Association and by an educational grant from Pharmacia & Upjohn, Inc. (Stockholm, Sweden).
Abbreviations: AER, Albumin excretion rate; AGE, advanced glycation end product; HbA1c, glycated hemoglobin; OR, odds ratio; VEGF, vascular endothelial growth factor.
Received November 9, 2000.
Accepted April 25, 2001.
References
This article has been cited by other articles:
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  A. Ashraf, G. Mick, S. Meleth, H. Abdullatif, X. Wang, and K. McCormick Effect of Insulin on Plasma Vascular Endothelial Growth Factor in Children with New-Onset Diabetes J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4920 - 4923. [Abstract] [Full Text] [PDF]
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 D. R. Cha, Y. S. Kang, S. Y. Han, Y. H. Jee, K. H. Han, J. Y. Han, Y. S. Kim, and N. H. Kim Vascular endothelial growth factor is increased during early stage of diabetic nephropathy in type II diabetic rats J. Endocrinol., October 1, 2004; 183(1): 183 - 194. [Abstract] [Full Text] [PDF]
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; those with
VEGF >160 pg/ml) and group 2 (
; those with VEGF <160 pg/ml).