Serum and Urinary Nitrites and Nitrates and Doppler Sonography in Children With Diabetes

We recruited 42 Caucasian children with type 1 diabetes (22 boys and 20 girls, aged 6–18 years) who attended the Diabetic Centre of the Department of Pediatrics, University of Chieti, Italy, between November 2002 and March 2003. All subjects were otherwise in good health; none had clinical or laboratory signs of kidney disease or microalbuminuria; none were affected by other complications of diabetes, such as retinopathy (evaluated by stereoscopic fundus photography) or neuropathy (evaluated by nervous conduction velocity and autonomic tests), or by other autoimmune disease. None of the patients were taking other drugs except for insulin. Daily insulin requirement was assessed. All subjects were assigned into the prepubertal or pubertal group on the basis of breast development in girls and genital development in boys, according to the criteria of Tanner. Prepubertal children were defined as Tanner stage 1 (10 boys and 10 girls, mean ± SD age 9.75 ± 2.02 years, mean duration of diabetes 5.50 ± 2.39 years) and pubertal as Tanner stages 2–5 (12 boys and 10 girls, mean age 14.63 ± 1.39 years, mean duration of diabetes 8.50 ± 3.30 years). We recruited, as a control group, 41 Caucasian children comparable for sex, age, and pubertal stage and divided them into prepubertal (11 boys and 10 girls, mean age 9.52 ± 1.94 years) and pubertal (10 boys and 10 girls, mean age 14.75 ± 1.29 years) who were admitted to the Department of Pediatrics of the University of Chieti, Italy, for minor diseases. Blood sampling, anthropometric measurements, and Doppler ultrasonographic evaluation was performed only after complete recovery of the disease. All patients, or their parents, gave informed consent. The study was approved by the ethics committee of the University of Chieti, Italy.

Body weight was measured with a digital scale to the nearest 0.1 kg, and height was measured in triplicate with a wall-mounted stadiometer. The BMI (weight in kilograms divided by the square of the height in meters) was assessed as the fatness index. Blood pressure was measured by auscultation with a standard mercury sphygmomanometer in a quiet and comfortable environment after 3–5 min of rest. The cuff size was appropriate for the size of the 40% of the arm circumference midway between the olecron and the acromion, according to the recommendations of the Task Force on Blood Pressure Control in Children (13).

Blood samples were collected in fasting conditions and immediately centrifuged at 4°C. Plasma glucose level was determined by using the glucose oxidase method. HbA_1c (A1C) concentrations were measured every 3 months using a high-performance liquid chromatography method (Diamant analyser; BioRad, Richmond, CA). The normal range was 4.2–6.0%, with an intrassay coefficient of variation (CV) of 3%. All the patients had at least three determinations per year, and the mean of these determinations was used for statistical analysis. The creatinine concentration was measured by an autoanalyzer (Astra 8; Beckam Instruments/Hybritech, Palo Alto, CA). Urinary albumin concentration was measured by double-antibody radioimmunoassay (Pharmacia, Uppsala, Sweden) with a sensitivity 0.5 mg/l, an intra-assay CV of 4.5%, and an interassay CV of 5.3% in the range of 10–50 mg/l. None of the children with diabetes had persistent microalbuminuria (defined as an albumin excretion rate >20 μg/min in two of three overnight urine collections in 6 months). Glomerular filtration rate (GFR) was assessed using radionuclide-imaging studies with ^99mtechnetium dimetiltriamino pentacetic acid, as previously described (14).

In the cell, NO undergoes a series of reactions with several molecules present in biological fluids and is eventually metabolized to nitrite (NO₂⁻) and nitrate (NO₃⁻). Thus, the best index of total NO production is the sum of both nitrite and nitrate, commonly quantified in a two-step assay (15). In this study, serum NO₂⁻+NO₃⁻ levels were measured in triplicate by the conversion of NO₃⁻ to NO₂⁻ by a commercially available kit based on the Griess reaction (active motif’s NO quantitation kit), following the manufacturer’s instructions. Blood samples were collected in fasting conditions, and serum nitrite (NO₂⁻) and nitrate (NO₃⁻) concentrations were measured as previously reported (16). NO urinary metabolites were evaluated in the overnight urine collection; for each patient, 50 μl antioxidant solution was added to 40 ml fresh overnight urine and frozen at −20°C. Nitrite (NO₂⁻) and nitrate (NO₃⁻) contents were evaluated with the nitrate reductase method.

The children’s eating habits reflect the typical Mediterranean diet. Consumption of foods containing nitrates (e.g., spinach, beets, cabbage, cauliflower, beetroot, and lettuce) was discouraged for the 48 h preceding the measurements to avoid the dietary influence of NO assay.

The measurements were commenced in patients fasting for at least 8 h, after recording pulse and blood pressure. All the examinations were performed using a 3.5–4.0 MHz vector array transducer, after 15 min rest in a horizontal position, as previously described (10). The first measurement was the size of the left and right kidney. The main trunk of the renal artery was displayed using color Doppler ultrasonography. Three measurements each were taken within 5 min in the vicinity of the interlobary artery at the boundary of the center of the kidney and the upper and lower pole using pulsed Doppler. The RI, according to Pourcelot, was calculated on the basis of the following formula: [(peak systolic velocity − peak diastolic velocity)/peak systolic velocity].

The average value of three bilateral measurements was taken for statistical analysis. All the examinations were performed twice by the same operator, without knowledge of the patient group (case or control).

All values are expressed as means ± SD. Differences in unpaired samples were examined by Mann-Whitney testing. A P value of 0.05 was considered statistically significant. Correlation between variables was evaluated using Pearson’s correlation coefficient. Data input and basic evaluation were carried out using the SPSS version 10.0 software.

Clinical characteristics of subjects are summarized in Tables 1 and 2. Children with and without diabetes, both prepubertal and pubertal, did not differ in sex, age, and BMI. Children with diabetes had significantly increased values of GFR compared with control subjects (144.94 ± 42.49 vs. 128.30 ± 15.94 ml/min, respectively, P = 0.005).

Serum and urinary NO concentrations were significantly increased in children with diabetes compared with control subjects (30.26 ± 6.52 vs. 24.47 ± 7.27 mmol/l, respectively, P = 0.001). In particular, prepubertal children with diabetes had significantly higher NO₂⁻+NO₃⁻ serum levels than prepubertal healthy children (30.88 ± 7.33 vs. 25.13 ± 8.06 mmol/l, P = 0.026); the same findings weree detected in the pubertal group (29.68 ± 5.79 vs. 23.74 ± 6.43 mmol/l, P = 0.012). Urinary NO₂⁻+NO₃⁻ levels were significantly higher in children with diabetes (345.07 ± 151.35 vs. 245.86 ± 80.25 mmol/l, P = 0.002) in both the prepubertal (296.47 ± 110.45 vs. 238.39 ± 59.37 mmol/l, P = 0.046) and pubertal (391.12 ± 172.31 vs. 296.85 ± 131.12 mmol/l, P = 0.013) groups.

Doppler RI was significantly higher in children with diabetes than in healthy control children (0.64 ± 0.03 vs. 0.60 ± 0.04, respectively, P = 0.035) in both the prepubertal (0.65 ± 0.04 vs. 0.61 ± 0.05, P = 0.014) and the pubertal (0.64 ± 0.03 vs. 0.60 ± 0.03, P = 0.002) groups.

In the diabetic group, a significant positive correlation between serum NO₂⁻+NO₃⁻ levels and urinary NO₂⁻+NO₃⁻ levels (P = 0.002, r = 0.374) was found. Serum NO₂⁻+NO₃⁻ concentrations also correlated positively with A1C (P = 0.004, r = 0.329), GFR (P = 0.05, r = 0.224), and Doppler RI (P = 0.032, r = 0.262). Urinary NO₂⁻+NO₃⁻ concentrations correlated positively with A1C (P = 0.001, r = 0.394) and GFR (P = 0.0001, r = 0.480). Doppler RI correlated positively with A1C (P = 0.000, r = 0.424) and diabetes duration (P = 0.020, r = 0.386) (Table 3). There were no correlations in the control group, neither for serum and urinary NO₂⁻+NO₃⁻ nor for Doppler RI values (Fig. 1). No sex differences in NO results or Doppler RI were found.

It has been clearly demonstrated that hyperglycemia is directly related to hyperfiltration and renal hyperperfusion (17), and it has been causally linked to vascular and glomerular dysfunction. There is no shortage of plausible biochemical mechanisms that could lead to functional changes and/or tissue damage. A glucose-dependent abnormality in NO production and action has recently been the subject of extensive investigation. Diabetes triggers mechanisms that in parallel enhance and suppress NO bioavailability in the kidney. During the early phase of nephropathy, the balance between these two opposing forces is shifted toward NO production. This plays a pivotal role in the development of characteristic hemodynamic changes and may contribute to consequent structural alteration in the glomeruli (18). Nonetheless, alterations of the NO system in the diabetic kidney and their role in the pathophysiology of diabetic nephropathy still represent a complex and controversial scenario; many important mechanisms can comodulate NO activity in a particular system (e.g., glycemic control, insulin treatment, duration of the disease, development of diabetes complications), and there are a number of topics in this area that warrant further investigation. In the present study, serum and urinary NO₂⁻+NO₃⁻ levels were found to be significantly increased in children with diabetes compared with healthy subjects and showed a significantly positive correlation with A1C and GFR. This was detected in prepubertal and pubertal children, supporting the hypothesis that enhanced NO production occurs at the earliest stages of diabetic nephropathy, before microalbuminuria appears, and is independent from diabetes duration and puberty. However, other investigators (19) found decreased levels of the same parameters. Puberty has been recognized as a major risk factor for the development of microangiopathy in children with diabetes, although it is not necessarily associated with the progression to frank proteinuria. It accelerates microvascular complications of diabetes, but prepubertal years of hyperglycemia appear to contribute to its development (20). The onset of microvascular complications can be presumed to start very early in the course of diabetes, perhaps at the disease onset, and the earliest stages can be often detected as soon as 2–5 years after diagnosis (21). Accordingly, in this study, both pubertal and prepubertal children with diabetes have higher NO values than healthy control subjects, suggesting no effect of puberty.

Histologically, basal membrane thickening seems to have the major predictive value to estimate the risk of disease progression (22), but it is unthinkable to implement invasive methods, such as renal biopsy, for screening purposes, especially in children. Doppler ultrasonography seems to be a reliable method for renal explorations by providing not only morphological but also physiological data with the perfusion study. The Doppler RI [(peak systolic velocity − peak diastolic velocity)/peak systolic velocity] has advanced as a useful parameter for quantifying the alteration in renal blood flow that may occur with renal disease. It reflects intrarenal vascular resistance, which is markedly linked to vascular compliance. Elevated RIs were reported with vascular-interstitial disease, including diabetic nephropathy (but not glomerulopathies); this may be due to the decreased tissue and vascular compliance and to the increased renal vascular resistance (23,24). Early functional and structural abnormalities present after a few years of diabetes might be responsible for the precocious alteration in renal hemodynamics, and this might be reflected in increased RI. Nonetheless, only few studies describing the application of Doppler ultrasonography in the evaluation of intrarenal hemodynamic abnormalities in diabetic nephropathy have been published, and most of them were performed in adults with type 1 or type 2 diabetes (9,10). Recently, a study evaluated the role of renal Doppler ultrasonography as a predictor of preclinical diabetic nephropathy in children and documented that ultrasonography is not useful for the prediction and detection of early diabetic nephropathy (25); however, there is no general agreement for the predictive value of Doppler ultrasonography in patients with diabetic nephropathy (12,25–28).

In the present study, we wanted to assess whether Doppler ultrasonography could be used to detect changes in renal RI in children with diabetes, the correlations between these parameters, and whether the increased NO excretion might exist. We observed that children with diabetes had significantly increased values of Doppler RI compared with healthy control subjects, although all the RI values (in diabetic and healthy children) were ≤0.70, which is actually accepted as the boundary normal RI value for healthy adults and children (29). This was found in the prepubertal and pubertal children with diabetes, all having normal renal function. Increased RI correlates positively with A1C, duration of diabetes, and NO excretion. No correlation between NO and RI was found in the control groups.

The significant positive association between NO and Doppler RI in children with diabetes suggests that precocious abnormality in NO production and action occurring early in the course of the disease may have contributed to the loss of maintaining the normal state of vascular tone and, thus, to the higher starting RI values. At the single-nephron level, diabetic hyperfiltration is characterized by disproportionately decreased afferent arteriolar resistance, resulting in elevated glomerular capillary pressure; this might be reflected in the increased RI. Considering the renal hemodynamic actions of NO, this substance is a good candidate for mediating diabetic hyperfiltration (18).

In conclusion, this study demonstrates that in children with diabetes, chronic hyperglycemia may act through a mechanism that involves increased NO production and/or action and contributes to generating intrarenal hemodynamic abnormalities, detectable by Doppler ultrasonography even in early diabetic nephropathy before microalbuminuria appears, and acts independently from duration of diabetes and puberty. Although our data have to be confirmed in further longitudinal studies, they suggest that evaluation of NO excretion and Doppler intrarenal RI may be a useful complementary test in the evaluation of early stages of diabetic kidney disease. Longitudinal monitoring of a panel of laboratory markers, such as those used in the current study, may better define their relevance in progressive kidney disease and provide greater insight into the mechanisms underlying this process.