Urinary N-Acetyl B Glucosaminidase as an Earlier Marker of Diabetic Nephropathy and Influence of Low-Dose Perindopril/Indapamide Combination

Abstract

Introduction. Tubulointerstitial injury is both a key feature of diabetic nephropathy and an important predictor of renal dysfunction. N-Acetyl B glucosaminidase (NAG) is derived from proximal tubular cells and is widely used to evaluate tubular renal function. Objective. The objective of this study is whether NAG can be used as an early marker of diabetic nephropathy by comparing the urinary NAG levels between healthy controls and diabetic patients and determining changes in urinary NAG excretion after treatment with low-dose combination perindopril (2 mg)/indapamide (0.625 mg)/o.d. Materials and Methods. A total of 50 patients (29 female) with type II diabetes mellitus applying to our diabetes outpatient clinics for the first time were included in our study (Group 1). Diabetic patients were classified into three subgroups on the basis of their duration of diabetes: Group 1_A (n = 15) ≤3 years, Group 1_B (n = 19) 3 to 5 years, and Group 1_C (n = 16) >5 years. The inclusion criteria were no prior use of antihypertensive agents; blood pressure <130/85 mmHg; urinary albumin excretion <30 mg/day; and absence of renal failure, diabetietes, and hypertensive retinopathy. A total of 30 healthy individuals (16 female) (Group 2) were assessed as the control group. Systolic and diastolic blood pressures, HbA1c, body mass index, 24-h microalbuminuria (MAU), and NAG measurements in urine samples were performed by using colorimetric assay method in an analyzer (Roche Cobas Mira). The assay defined as fragmentation of 3-cresolsulfonphthaleinyl-N-acetyl-β-D-glucosaminide molecule by NAG to 3-cresolsulphonphthalein and N-acetylglucosamine molecules and serum creatinine were measured in all groups. Type II diabetic patients were administered perindopril (2 mg)/indapamide (0.625 mg) combination once daily for 4 months, and urinary NAG levels were measured at the end of treatment. Results. Statistically significant differences were observed between the groups 1 and 2 with respect to the levels of NAG and HbA1c (p < 0.05). In the treatment group, NAG levels decreased significantly (p < 0.05), whereas blood pressure and HbA1c levels did not change significantly (p > 0.05). In diabetic patients, pretreatment NAG were lowest in Group 1_A and highest in Group 1_C, although the difference between the treatment subgroups was not statistically significant (p > 0.05). Conclusion. Urinary NAG excretion is elevated in type II diabetic patients as compared with the healthy individuals. Perindopril/indapamide administration is effective in reducing urinary NAG excretion in these patients, and this effect seems to be independent from blood pressure and glycemia control. Presence of tubular proteinuria may be an early indicator of diabetic renal disease in patients without microalbuminuria. Perindopril (2 mg)/indapamide (0.625 mg)/o.d. treatment may have beneficial effect on the tubulointerstitial damage in diabetic kidney disease.

Keywords:

• urinary NAG
• earlier marker
• diabetic nephropathy
• perindopril/indapamide combination

INTRODUCTION

Tubulointerstitial injury is both a key feature of diabetic nephropathy and an important predictor of renal dysfunction.[1]

Diabetic patients excrete several tubular markers in the urine, including lysosomal and brush border enzymes. N-acetyl B glucosaminidase (NAG) is a lysosomal enzyme that is derived from proximal tubular cells. Under normal circumstances it is not filtrated in the glomeruli, but its excretion increases in conditions associated with marked tubular injury. Thus, although proteinuria is commonly used to evaluate glomerular injury, measurement of urinary NAG is also widely used to assess tubular renal functions.[2],[3]

It is well established that the detection of microalbuminuria in a patient with diabetes mellitus indicates the presence of glomerular involvement in early renal damage. Recent studies have demonstrated that there is also a tubular component to renal complications of diabetes, as shown by the detection of renal tubular proteins and enzymes in the urine. In fact, tubular involvement may precede glomerular involvement because several of these tubular proteins and enzymes are detectable even before the appearance of microalbuminuria.[4]

Renal alterations become irreversible when clinical proteinuria occurs in diabetic patients and diabetic nephropathy progresses to chronic renal failure. Therefore, several studies have suggested a potential role for urinary NAG levels in predicting the incipient diabetic nephropathy. At present, however, there are few reports examining the early stage of diabetic nephropathy in type II diabetes.[4–6]

OBJECTIVES

1. To determine whether NAG can be used as an early marker for diabetic nephropathy by comparing urinary NAG levels in healthy individuals and diabetic patients

2. To determine the change in urinary NAG excretion in diabetic following treatment with perindopril/indapamide

MATERIALS AND METHODS

A total of 50 patients (29 female) with type II diabetes mellitus were included in the study (Group 1). The inclusion criteria were as follows:

• No use of OADs, insulin, antihypertensive agents, or lipid-lowering agents in the previous 3 months

• BP < 130/85 mmHg

• Urinary protein excretion <30 mg/day

• No diabetic or hypertensive retinopathy

• No urinary tract infection

• Absence of renal failure

• No significant history of cardiovascular disease (cardiac, cerebral, peripheric, vascular)

A total of 30 healthy controls (16 female) for comparison with baseline values were included in the study as the control group (Group 2).

Diabetic patients were classified on the basis of the duration diabetes: Group 1_A (n = 15) ≤3 years, Group 1_B (n = 19) 3 to 5 years, (n = 16) Group 1_C ≥5 years.

Systolic and diastolic blood pressure, HbA1c (immunoturbidimetric), 24-h microalbuminuria, NAG in spot urine sample and serum creatinine (by spectrophotometric assay), serum creatinine clearance (Cockcroft-Gault formula = (140 − age) × kg/72 × creatinine, and (BMI) body mass index (kg/boy2) were measured in both groups. Type II diabetic patients were treated with perindopril (2 mg)/indapamide (0.625 mg) tablets taken once daily for 4 months, and urinary NAG levels were measured. P values of 0.05 or less were considered, to be significant.

All data are expressed as mean ± SD. Student's t test for paired variables and chi-square test for the differences in categorical data were used. The difference was considered significant if the p value was <0.05.

RESULTS

There were no significant relation between NAG and HbA1c, MAU, age, creatinine, glomenular filtration rate (GFR), fasting blood glucose, insulin, HOMA, systolic blood pressure, diastolic blood pressure, and BMI measurements (p > 0.05).

Groups demonstrated statistically difference in terms of NAG and HbA1c levels (p < 0.05); however, there was no statistically difference in age, BMI, MAU, plasma creatinine, or blood pressure of the patients (p > 0.05) (Table 1).

Table 1 Pretreatment characteristics; comparison of groups

	Study group (n:50)	Control group (n:24)	p
Age (years)	53.93 ± 16.54	57.50 ± 5.93	>0.05
BMI (kg/m^2)	28.27 ± 4.3	28.36 ± 4.24	>0.05
Pretreatment NAG (U/L)	7.90 ± 7.06	4.15 ± 1.93	<0.05
Pretreatment HbA1c (%)	8.36 ± 1.97	4.95 ± 0.70	<0.0001
MAU (mg/day)	11.70 ± 8.26	10.94 ± 8.31	>0.05
Plasma creatinine (mg/dL)	0.84 ± .18	0.92 ± 0.16	>0.05
Systolic blood pressure (mmHg)	115.4 ± 8.6	113.9 ± 8.1	>0.05
Diastolic blood pressure (mmHg)	74.7 ± 6.6	73.9 ± 9.2	>0.05

There was a significant decline in NAG levels in the treated group (p < 0.001); HbA1c levels increased, although this was not statistically significant (p > 0.05) (Table 2). No statistically significant difference was observed among all groups concerning pre- and posttreatment blood pressures (p > 0.05) (Table 3).

Table 2 Comparison of baseline and posttreatment values

	Pretreatment	Posttreatment	t	p
NAG (U/L)	7.90 ± 7.06	5.59 ± 3.97	3.43	<0.001
HbA1c (%)	8.36 ± 1.97	9.82 ± 14.76	−0.72	NS

Table 3 Pre- and posttreatment blood pressure values

Duration of diabetes (years)	Pretreatment SBP	Posttreatment SBP	Pretreatment DBP	Posttreatment DBP
Group 1A ≤3	113.3 ± 8.4	112.5 ± 6.0	74.2 ± 7.7	73.4 ± 7.2
Group 1B 3–5	116.2 ± 4.4	114.9 ± 7.6	74.4 ± 6.1	74.2 ± 5.8
Group 1C ≥5	116.1 ± 5.6	116.3 ± 8.5	76.1 ± 5.5	75.3 ± 5.6

The pretreatment NAG levels in groups classified according to the duration of diabetes were lowest in Group 1_A and highest in Group 1_C, but the differences between the groups were not significant (p > 0.05), and a positive correlation between NAG levels and the duration of diabetes was observed (Table 4).

Table 4 Correlation of duration of diabetes and NAG levels

NAG levels (U/L)	Pretreatment	Posttreatment
Age group 1A ≤3	6.43 ± 6.61	4.07 ± 3.51
Age group 1B 3–5	7.56 ± 6.49	6.54 ± 3.60
Age group 1C ≥5	8.33 ± 6.30	6.28 ± 4.40
F	0.38	2.04
P	>0.05	>0.05

*p A vs. B, A vs. C, B vs. C groups.

DISCUSSION

Although NAG exists in physiological levels in the urine of healthy individuals, its excretion increases in significant tubular lesions. Therefore, although proteinuria is used as a marker a marker of glomerular injury, urinary NAG is also widely to gauge the tubular renal function.[2],[3],[7]

Our study shows that NAG, a lysosomal enzyme normally excreted in low amounts in urine as a consequence of the physiological exocytosis process in proximal tubular cells, is excreted in significantly greater amount in patients with type II diabetes mellitus compared with healthy subjects. The difference between the two groups was significant (p < 0.05).

Perez-Blanco et al. showed that urinary NAG excretion was elevated in patients with type II diabetes mellitus with no clinical or analytical signs of nephropathy and in patients with evident diabetic nephropathy with respect to healthy controls.[8]

Fujita et al. demonstrated a higher level of urinary NAG excretion in 22 patients with impaired glucose tolerance (IGT) and normal blood pressure when compared with 41 healthy subjects. It is believed that increased urinary excretion of NAG in IGT may be due to the adverse effect of elevated levels of plasma proteins in IGT, which are highly filtered through the glomerular capillary on tubular cells.[9]

Whether this isolated tubular dysfunction constitutes a precursor of incipient diabetic nephropathy has not yet been established. It has been shown that abnormal urinary NAG excretion has a high positive predictive value for the development of microalbuminuria.[5]

In the phase of incipient diabetic nephropathy, increased glomerular hyperfiltration induces the mesangial expansion and high tendency for urinary albumin excretion to increase. In these patients, hyperglycemia causes an imbalance between oxidation/antioxidation systems that occurs with increased production and decreased clearance of reactive oxygen species, as well as with a dysfunctional antioxidant defense system. In addition, hyperglycemia results in elevated angiotensin II levels that are vasosoactive peptid. Angiotensin II stimulates proliferation of mesangial synthesis of TNF-β that is a profibrogenic factor. Rising in both glomerular pressure and TNF-β levels by angiotensin II induces overt proteinuria and renal fibrogenic processes.[10]

Brenner et al. showed that ACE inhibitors exert renoprotective effect through intrarenal change, including a decrease in the intraglomerular pressure. These researchers suggested that this favorable effect is likely to be caused by the suppression of transforming growth factor-β(TGF-β)-dependent pathways in both the glomeruli and tubulointerstitium.[10],[11]

Ravid et al. proposed that ACE inhibitors can prevent the development of microalbuminuria in normotensive normoalbuminuric type II diabetic patients.[12]

Our study administration of perindopril (2 mg)/indapamide (0.625 mg)/o.d. tablets to patients with type II diabetes milletus elevated urinary NAG levels produced a significant decrease NAG excretion. This decrease is independent from blood pressure, HbA1c levels, serum creatinine, GFR, or BMI.

Studies have shown that in patients with absence of any signs of renal dysfunction, including microalbuminuria, urinary NAG activity starts at around the third year of type II diabetes milletus. In multiple regression analysis, irrespective of protein levels, NAG was found to be an independent correlate.[13]

Although we failed to show a significant correlation between the duration of diabetes and the levels of NAG (P > 0.05) in diabetic patients, pretreatment NAG were lowest in group A and highest in group C (Table 4).

Clinical evidence suggesting an important role for microalbuminuria in defining patients with cardiovascular disease (CVD) risk is increasing. In fact, CVD is the leading cause of death in patients with microalbuminuria. Although the underlying mechanisms are far from being clear, it is generally accepted that microalbuminuria reflects the extensive endothelial dysfunction that can pave the way for both CVD and renal disease. Therefore, to reduce the risk of CVD and renal failure, markers that can predict the likelihood of renal dysfunction at an earlier stage of microalbuminuria are required.

In conclusion, urinary NAG excretion is elevated in type II diabetic patients as compared with healthy individuals. Presence of tubular proteinuria may be an early indicator of diabetic renal disease in patients without microalbuminuria.

Perindopril (2 mg)/indapamide (0.625 mg)/o.d. administration is effective in reducing urinary NAG excretion in these patients, and this effect seems to be independent of blood pressure and glycemia control. It is possible to hypothesize that perindopril (2 mg)/indapamide (0.625 mg)/o.d. may have beneficial effects on the tubulointerstitial damage in diabetic kidney disease.