S. Abdel Shakour,1 H. El-Hefnawy,1 M. Y. El-Yamani1 and Y. Azmi1tc "S. Abdel Shakour,1 H. El-Hefnawy,1 M. Y. El-Yamani1 and Y. Azmi1"

الاستفادة من N-أسيتيل-بيتا-D - غلوكوز أمينيداز (النَّاغ NAG) في بول الأطفال السكَّريـين كواسم باكر للاعتلال الكلوي السكَّري

سامح عبد الشكور، محمد هشام الحفناوي، محمد يحيى اليماني وياسر عزمي صقر

الخلاصـة: تم استقصاء أسيتيل غلوكوز أمينيداز (ناغ) في بول الأطفال السكَّريـين المصابين بالسكَّري من النمط الأول باعتباره واسماً باكراً للإصابة بالتخرُّب النُبَيْي، ودراسة علاقته ببيلة المكروألبومين وبالسيطرة على سكر الدم. وقد تم تعيين نسبة النَّاغ NAG إلى الكرياتينين، وبيلة المكروألبومين وغليكوزيل الهيموغلوبين Hb A1، وبيلة المكروألبومين وقِيَم ضغط الدم الانقباضي والانبساطي. وقد وُجد أن 59.5% من الأطفال السكَّريين لديهم ناغ NAG إيجابي في البول، وأن 38.1% منهم لديهم بيلة مكروألبومينية إيجابية

ABSTRACT We investigated urinary N-acetyl-beta-D--glucosaminidase (NAG) levels in children with type 1 diabetes as an early marker of tubular damage and studied its correlation with microalbuminuria and glycaemic control. The study group comprised 42 children with type 1 diabetes and 20 healthy children as control. Urinary NAG to urinary creatinine ratio, micro­albuminuria, glycated haemoglobin (Hb A1c), blood urea and serum creatinine were estimated. Urinary NAG levels in the children with diabetes were significantly higher than those of controls. There were positive correlations between urinary NAG levels and microalbuminuria, Hb A1c and systolic and diastolic blood pressure values. We found that 59.5% of diabetic children were positive for urinary NAG, while 38.1% of them were positive for microalbuminuria.

La N-acétyl-bD glucosaminidase (NAG) urinaire chez des enfants diabétiques en tant que marqueur précoce de la néphropathie diabétique

RESUME Nous avons examiné les taux de NAG urinaire chez des enfants atteints de diabète de type I en tant que marqueur précoce de lésion tubulaire et avons étudié sa corrélation avec la microalbuminurie et le contrôle glycémique. Le groupe étudié comprenait 42 enfants atteints de diabète de type I et 20 enfants en bonne santé en tant que témoins. Le rapport N-acétyl-bD glucosaminidase urinaire-créatinine urinaire, la microalbuminurie, l'hémoglobine glycosylée (HbA1c), l'urée sanguine et la créatininémie ont été estimés. Les taux de NAG urinaire chez les enfants diabétiques étaient significativement plus élevés que chez les témoins. Il y avait une corrélation positive entre les taux de N-acétyl-bD glucosaminidase urinaire et la microalbuminurie, entre l'hémoglobine glycosylée et les valeurs de la pression artérielle systolique et diastolique. Nous avons trouvé que 59,5 % des enfants diabétiques étaient positifs pour la N-acétyl-bD glucosaminidase urinaire, tandis que 38,1 % d'entre eux étaient positifs pour la microalbuminurie.

Introduction

Diabetic microangiopathy denotes wide­spread microvascular abnormalities that occur frequently in longstanding diabetes and affect many body organs. In renal affection, the lesions involve glomerular capillaries, as well as the afferent and efferent arterioles. Afferent arterioles usually exhibit gross thickening of their walls. The renal tubules show similar changes to those found in persistent glome­rular nephritis, and very occasionally the proximal tubules contain large quantities of glycogen [1]. It was proven by Yaqoob et al. in 1995 that diabetic nephropathy is associated with comparable degrees of increased serum creatinine and albuminuria as markers of glomerular damage [2]. In addition, increased urinary excretion of leucine aminopeptidase, N-acetylglutamyl transferase, retinol binding protein and beta2-microglobulin as markers of tubular damage. Moreover, the measurement of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme localized in the proximal tubules, served as an additional parameter for the assessment of tubular damage [3].

NAG is a lysosomal enzyme originating in the renal tubules, and its urinary excre­tion is elevated in various renal disorders. Gibb et al. suggested that the tubular abnormalities are present early in the course of type 1 diabetes and that the early increase in urinary excretion of albu­min may be, at least partly, tubular in origin and may be influenced by glycaemic cont­rol [4]. He also suggested that elevated urinary transferrin and NAG excretion rates may precede elevation of albumin excretion in children with type 1 diabetes.

Methods

The aim of this study was to investigate the level of urinary NAG in children with diabetes in comparison with urinary mic­roal-bumin as an early marker of diabetic nephropathy and to study their correlation with glycaemic control.

The study was carried out on 42 child­ren with type 1 diabetes mellitus who attended the paediatric clinic of the Natio­nal Institute of Diabetes and Endocrinology in Cairo, Egypt (NIDE). Their ages ranged between 5 and 15 years, and there were 22 females and 20 males.

Another 20 healthy children of the same age range were included in the study as a control group. There were 11 females and 9 males.

Both groups were subjected to the following:

•    Full history taking, including duration of diabetes, methods of treatment, daily insulin requirements and any symptoms or signs of diabetic complications.

•    Complete physical examination, both general and systemic, and observation for any manifestations of complica­tions. Particular attention was given to blood pressure measurement.

•    Laboratory investigations, which inclu­ded:

•   complete blood picture

•   blood urea and serum creatinine

•   fasting plasma glucose.

Glycated haemoglobin (Hb A1c) was estimated using the chromatography me­thod on a DCA 2000 apparatus [5]. The ratio of urinary NAG index in random morning urine sample was estimated. This ratio is considered as an indicator for urinary NAG level in 24 hour urine. The upper  reference  limit for NAG was 8.15 U/g cr using the colorimetric technique [5].

The ratio of urinary albumin in mg to urinary creatinine (cr) in g (albumin index) in a random morning urine sample was estimated and considered an indicator for microalbumin level in a 24 hour urine collection. Children were classified as normoalbuminuria (if urinary albumin < 30 mg/g cr), microalbuminuria (if urinary albumin fell between 30–300 mg/g cr) and macroalbuminuria (if urinary albumin > 300 mg/g cr). This was done using the chromatographic DCA 2000 method [5].

To exclude any causes other than dia-betes that might be responsible for renal abnormalities, all children positive for microalbuminuria or NAG excretion were subjected to urinalysis with culture and sensitivity and abdomino-pelvic ultraso­nography.

Statistical analysis of the results was carried out using standard computer prog­rams.

Results

Urinary NAG levels in the diabetic group (42 patients) ranged between 2.6 and 80.4 U/g cr (mean = 19.9 ± 18.9 U/g cr). Of these, 25 patients showed positive figures for NAG excretion (59.5%) (Figure 1). Urinary microalbuminuria was positive in 16 patients only (38.1%), all belonging to the NAG-positive group. The microalbu­min level in the children positive for NAG excretion ranged from 5.8 to 120.8 mg/g cr (mean = 23.4 ± 25.3 mg/g cr).

Figure 1 Percentage of children with diabetes with positive urinary NAG and those with positive microalbuminuria in the patient group under study
 

Table 1 shows that there was no sig­nificant difference found between the children with diabetes and the control children regarding age, sex, systolic and diastolic blood pressure values, blood urea, serum creatinine, serum cholesterol and serum triglyceride levels (P > 0.05). How­ever, a significant difference was found between both groups as regards urinary NAG levels (mean = 19.9 ± 18.9 U/g cr for cases and 3.66 ± 2.59 U/g cr for controls) (P < 0.01). There was also a significant difference (P < 0.01) found with  respect to microalbumin levels in both groups (mean = 23.43 ± 25.32 mg/g cr for cases and 10.23 ± 5.12 mg/g cr for controls) and to glycated Hb A1c (mean = 8.24 ± 2.05% for cases and 5.19 ± 0.67% for controls) (P < 0.01).

Table 1 Characteristics and blood and urine measurements of children with diabetes compared with control children

Table 2 shows the presence of positive correlation between urinary NAG levels in the children with diabetes and other para­meters, namely the level of urinary microal­bumin (P < 0.01), glycated Hb A1c (P < 0.01), duration of diabetes (P < 0.01), and systolic and diastolic blood pressure values (P < 0.05). No correlation (P > 0.05) was found between urinary NAG levels in diabetics and age, blood urea, serum crea­tinine, serum cholesterol, serum trigly­cerides and fasting plasma glucose levels.

Table 2 Correlation (r) of urinary NAG levels in children with diabetes to different factors

Discussion

We investigated the level of urinary NAG and urinary microalbumin in children with diabetes. Urinary NAG was regarded as an indicator of tubular affection, while micro­albuminuria denoted glomerular affection. The analytes were studied in respect to their sensitivity in detecting early renal affection in the study group. In addition, the correlation of these analytes to gly­caemic control was investigated.

The results of this study showed that 59.5% of the children with diabetes were positive for urinary NAG levels, while only 38.1% of them were positive for micro­albuminuria. These latter patients were a subgroup of those patients found to be positive for urinary NAG levels. In ad­dition, a positive correlation between urinary NAG levels and microalbuminuria (P < 0.01) on the one hand, and Hb A1c (P < 0.01) on the other hand was clearly identified.

Many studies have investigated the is­sue of nephropathy among diabetic pa­tients, trying to understand the sequence of pathological events and to discover sensi­tive markers for early renal involvement. One feature of diabetic microangiopathy is endothelial dysfunction [6,7], which in type 1 diabetes precedes the onset of microalbuminuria [8].

Hsiao in 1996 examined 31 children with diabetes without any clinical evidence of nephropathy for urinary NAG and microalbuminuria [9]. He concluded that urinary NAG is a sensitive indicator of renal tubular injury (due to any cause or disease) and may be increased in children with diabetes even before the onset of mic­roalbuminuria. Also, the urinary NAG was significantly correlated with plasma Hb A1c levels.

In 1996, Platonova et al. studied the urinary enzymes in type 1 diabetes and reported that the degree of epithelial cell lesions in the renal tubules could be asses­sed from the urinary activities of enzymes at various sites such as NAG which could be an early marker of proximal nephritic tubules in type 1 diabetes [10].

In 1997, Caliskan et al. concluded that urinary NAG levels in children with dia­betes are significantly correlated with mic­roalbuminuria and Hb A1c [11]. They also concluded that tubular dysfunction and/or damage occurs in type 1 diabetes earlier than glomerular affection.

Moreover, in 1997, Hirai M et al. conc­luded from a study on people with type 2 diabetes that the NAG index and albumin index in random spot urine samples may serve as early functional indicators of diabetic nephropathy in people with dia­betes [5]. They found that the NAG index positively correlated with systolic blood pressure, duration of diabetes, Hb A1c and albumin index. The study was done, how­ever, on individuals with type 2.

In 1998, Kordonouri et al. reported the findings of a 7-year follow-up study of children and adolescents with type 1 dia­betes with normal albumin excretion rate [12]. They maintained that urinary NAG excretion could develop before microalbu­minuria. The investigators observed that patients with consistently low NAG levels did not progress to microalbuminuria. In contrast, patients with increased NAG levels were found to develop microalbu­minuria at a later stage.

In 1999, Uehara et al., in an experimen­tal study on diabetic rats, noticed that changes in glucose metabolism were as-sociated with proteinuria and an increase in urinary excretion of NAG [13]. Further­more, in 1999, Kordonouri et al. investiga­ted the prevalence of incipient renal dysfunction in two groups with identical duration of type 1 diabetes but with either childhood or adult onset of the disease [14]. The pattern of glomerular (albumin) and tubular (alpha1-microglobulin and NAG) urinary protein excretion was stu­died in 97 patients. The results suggested that there was no difference concerning the prevalence of incipient diabetic glomeru­lopathy between both groups. However, a more frequent impairment of tubular func­tion was observed in young patients with diabetes onset in childhood. The investiga­tors attributed this finding to non-optimal glycaemic control in this population. Pa­tients with diabetes onset in childhood showed significantly higher excretion of NAG compared to those with diabetes onset after the age of 16 years. The excre­tion of tubular markers correlated signifi­cantly with Hb A1c values in both groups. In multiple regression analysis, tubular proteinuria and diabetes duration correlated significantly to microalbuminuria.

It is obvious from all of the above that our results stand in agreement with most of the results of other investigators. Thus, we can conclude that tubular dysfunction occurs earlier than glomerular dysfunction in children with diabetes. Positive correla­tion of urinary NAG levels with glycaemic control was established. The finding that urinary NAG is an earlier marker of neph­ropathy than microalbuminuria makes it feasible to recommend that all children with diabetes have urinary NAG levels measured regularly for early detection of diabetic nephropathy.

References

1.     Davison AM. Diabetes mellitus and other metabolic disorders. In: MacLeod J, Edwards C, Bouchier I, eds. David­son’s principles and practice of medi­cine. Toronto, Churchill Livingstone, 1987:481.

2.     Yaqoob M et al. Tubular damage in microalbuminuric patients with primary glomerulonephritis and diabetic nephro­pathy. Journal of renal failure, 1995, 17(10):43–9.

3.     Andersen AR et al. Diabetic nephro­pathy in type 1 (insulin dependent) diabetes: an epidemiological study. Dia­betologia, 1983, 25:496–501.

4.     Gibb DM et al. Renal tubular proteinuria and microalbuminuria in diabetic pa­tients. Archives of disease in childhood, 1989, 64(1):129–34.

5.     Hirai M et al. [Microalbumin and N-acetyl-beta-D-glucosaminidase in ran­dom spot urine samples as predictors of diabetic nephropathy in elderly non-insulin dependent diabetic patients.] Nippon ika daigaku zasski [Journal of Nippon Medical School], 1997, 64(6): 518–25 [in Japanese].

6.     Haller H. Endothelial function, general considerations. Drugs, 1997, 53(suppl. 1):1–10.

7.     La Selva M et al. The role of endothelium in the pathogenesis of diabetic micro­angiopathy. Acta diabetologia, 1993, 30:190–200.

8.     Stehouwer CD et al. Endothelial dys­function precedes development of mic­roalbuminuria in IDDM. Diabetes, 1995, 44:561–4.

9.     Hsiao PH et al. Urinary N-acetyl-beta-D-glucosaminidase activity in children with IDDM. American journal of nephrology, 1996, 16(4):300–3.

10.  Platonova LV et al. K voprosu o fermen­turii pri insulinzavisimom sakharnom diabete. [Urinary enzymes in IDDM.] Voprosy meditsinskoi khimii, 1996, 42(1):70–6.

11.  Caliskan S et al. Tubular markers in children with IDDM. Turkish journal of pediatrics, 1997, 39(2):213–8.

12.  Uehara Y et al. Angiotensin II subtype-1 receptor antagonists improve hemo­dynamic and renal changes without af­fecting glucose metabolisms in genetic rat model of non-IDDM. American jour­nal of hypertension, 1999, 12:21–7.

13.  Kordonouri O et al. Predictive value of tubular markers for the development of microalbuminuria in adolescents with diabetes. Hormone research, 1998, 1:32–7.

14.  Kordonouri O et al. The prevalence of incipient tubular dysfunction, but not of glomerular dysfunction, is increased in patients with diabetes onset in child­hood. Journal of diabetes complications, 1999, 13(5-6):320–4.