Diagnostic Value of the Aminopeptidase N, N-Acetyl-β-D-Glucosaminidase and Dipeptidylpeptidase IV in Evaluating Tubular Dysfunction in Patients with Glomerulopathies

Abstract

Aim. The aim of the present study was to investigate the value of the urine cell glycoprotein 1 (PC-1), aminopeptidase N (APN), N-acetyl-β-D-glucosaminidase (NAGA), and dipeptidylpeptidase IV (DPP IV) in the evaluation of tubular damage in patients with primary glomerulonephritis, diabetic nephropathy, and lupus nephritis. Subjects and Methods. PC-1, APN, NAGA, and DPP IV activities were determined in serum, urine, and lymphocytes of 178 subjects, including 10 patients with membranous nephropathy, 38 with IgA nephropathy, 29 with lupus nephritis, 51 with diabetic nephropathy, and 50 control subjects. Results. Urinary PC-1 excretion in IgA nephropathy group was significantly higher (p < 0.05) than in controls. Urinary NAGA excretion was markedly (p < 0.01) higher in membranous nephropathy group, and APN excretion in diabetic nephropathy group was significantly higher (p < 0.01) than in healthy controls. Urinary APN activity was significantly (p < 0.01) higher in both type 1 and type 2 diabetic patients with microalbuminuria, as well as urinary NAGA and DPP IV activities in type 2 diabetics with microalbuminuria (p < 0.01 and p < 0.05, respectively) compared to controls. Serum PC-1 and APN activities were significantly higher than the control level in membranous nephropathy group, as well as serum PC-1 and DPP IV activities in IgA nephropathy patients (p < 0.05). However, significantly lower serum DPP IV and APN activity was observed in type 2 diabetics with microalbuminuria compared to controls (p < 0.05). Conclusion. Damage of tubules in primary glomerulonephritis, lupus nephritis, and diabetic nephropathy is accompanied by a release of several tubular enzymes, with possible diagnostic and prognostic significance. Increased serum PC-1, APN, and DPP IV activities could be also of diagnostic significance.

Keywords:

• glomerulonephritis
• plasma cell glycoprotein 1
• aminopeptidase N
• N-acetil-β-D-glucosaminidase
• dipeptidylpeptidase IV

INTRODUCTION

Glomerulopathies are the leading cause of end-stage renal failure in the majority of kidney disease patients from Serbia. Studies of the outcome of different forms of progressive glomerulonephritis show that a major factor, apparently determining the outcome, is the presence and severity of tubulointerstitial changes, and not the degree of glomerular alteration.[1] Moreover, at the time of biopsy, tubulointerstitial changes correlate much better with the glomerular filtration rate. Proximal tubule-derived enzymes have been already recognized, in morphologically different forms of glomerulopathies, as valuable tools in evaluating the tubular damage, even in patients with normal renal function and normal urinary albumin excretion rate. However, none of those markers has been recognized as a marker offering the possibility to be therapeutically modified in order to slow down progression of the disease.

Plasma cell glycoprotein 1 (PC-1), known as ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), is a class II transmembrane glycoprotein, implicated in the pathogenesis of insulin resistance in obesity, diabetes, and uremia,[2] as it inhibited insulin receptor signaling[3] either at the level of the insulin receptor tyrosine kinase[4] or downstream at a post-receptor cite.[5] Urinary PC-1 was found to be mainly produced by the kidney.[6] An ectonucleotide pyrophosphatase has been found in the brush border of the proximal tubule; however, a highly active phosphodiesterase I was demonstrated in glomerular epithelial and mesangial cells.[7],[8] Its increased urinary excretion has been observed in newly diagnosed type 1 diabetic patients with poor glycemic control, though there is decreased excretion in type 1 diabetics with micro- or macroalbuminuria, in patients with primary glomerulonephritis, including those with renal failure, as well as in those without an apparent kidney damage.[9]

Aminopeptidase N (APN) is an exopeptidase with wide substrate specificity, widely expressed in numerous human cells and tissues.[10–12] However, its urinary excretion is an established marker of the damage of a brush border of the proximal tubule. Compared to healthy controls, urinary APN and N-acetyl-beta-D-glucosaminidase (NAGA) were found significantly elevated in GN patients. APN indicates very early tubular impairment, and, in some cases, APN is elevated even though NAGA is still within normal ranges.[13]

N-acetyl-beta-D-glucosaminidase is a lysosomal enzyme, clearly indicated as a valuable measure to evaluate tubular damage in kidney disease patients, even in the early stages. Urinary NAGA originates in renal proximal tubular cells and positively correlated with microalbuminuria and metabolic control. It was found to be abnormally raised in 60% of type 1 diabetics before any increase in albumin excretion rate. However, in type 2 diabetics, NAGA began to rise in the third year of diabetes, maintained a plateau between 3 and 10 years, and rapidly increased after the tenth year of the duration of this disease.[14],[15]

Dipeptydilpeptidase IV (DPP IV) is an intrinsic membrane glycoprotein, localized on glomerular visceral epithelial cells, endothelial cells, and the proximal tubule brush border.[16] DPP IV is a ubiquitous, multifunctional, serine protease enzyme and receptor with roles in the control of endocrine and immune function, cell metabolism, growth and adhesion. As an enzyme, DPP IV cleaves the N-terminal dipeptide from the incretin hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. This inactivates the hormones, thereby canceling their prandial insulinotropic effect.[17] One approach to restore incretin activity as a therapy for Type 2 diabetes has been the development of DPP IV inhibitors. Inhibitors of DPP IV have shown efficacy and tolerability when used to control the hyperglycemia of noninsulin-dependent animal models and human Type 2 diabetes.

The aim of the present study was to investigate the value of the PC-1 and several other renal proximal tubule-derived enzymes in evaluating tubular dysfunction in patients with primary glomerulopathies, diabetic nephropathy and lupus nephritis in order to suggest the possible therapeutic modification of their expression as a beneficial treatment in patients with glomerulopathies.

SUBJECTS AND METHODS

Subjects

The present study was carried out at the Institute of Nephrology and Hemodialysis, Faculty of Medicine, Nis, Serbia. Out of a total of 128 involved patients, recruited from hospitals and outpatient clinics, 10 patients suffered from membranous nephropathy (age 59.6 ± 7.4 years), 38 from IgA nephropathy (age 51.6 ± 12.2 years), and 29 from lupus nephritis (aged 43.0 ± 10.5 years), while 51 had diabetic nephropathy (aged 56.4 ± 12.5 years), including 9 type 1 diabetics and 42 patients with type 2 diabetes mellitus. The control group consisted of 50 healthy, unrelated subjects, aged 47.7 ± 11.6 years, with no personal history or first-degree relatives with kidney diseases or abnormal laboratory test results of clinical significance. The study was approved by the local Research Ethics Committee, and informed consent was obtained from all participants enrolled in the study.

Methods

Baseline Assessments

Blood samples and urine were taken after an overnight fast of 12 hours, and baseline biochemical analyses were performed on BioSystems S.A. (Costa Brava, Barcelona, Spain) using standardized protocols.

Urinary and Serum Enzyme Activities

Phosphodiesterase activity of plasma cell membrane glycoprotein 1 (PC-1) was measured by the hydrolysis of thymidine-5′-monophosphate p-nitrophenol ester (Sigma Chemical Co., St. Louis, Missouri, USA).[18] Aminopeptidase N (APN), N-acetyl-β-D-glucosaminidase (NAGA) and dipeptidylpeptidase IV (DPP IV) activities were determined by the spectrophotometric method, using alanine-p-nitroanilide, N-acetyl-β-D-glucosaminide, and p-nitroanilide as substrates, respectively.[9],[19],[20] Urinary enzyme activities were expressed as enzyme-to-creatinine ratios.

Lymphocyte Enzyme Activities

Peripheral blood mononuclear cells were isolated from 10 ml of freshly drown heparinized (50 IU/mL) blood, layered over Ficoll-Hypaque (Lymphoprep, Nyegard, Oslo, Norway), washed twice in RPMI 1640 (Flow Laboratories, Irvine, UK) culture medium containing 25 mM HEPES, 2 mM glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL), and resuspended at a concentration of 2 × 10⁶/mL in the same medium supplemented with 10% fetal calf serum. Peripheral blood mononuclear cells were incubated for 48h at 37°C in an atmosphere of 95% air and 5% CO₂. Non-adhering cells from the culture plates were transferred to centrifuge tubes after appropriate washing with saline. PC-1 (alkaline phosphodiesterase I, APD) activity was determined in 50 mM Tris-HCl buffer, pH 8.0, 130 mM NaCl, 1 mM MgCl₂, with 1.5 mM p-nitrophenol thymidine 5′-phosphate as a substrate. Incubation was carried out at 37°C for 3–10 min with gentle agitation under zero-order kinetic conditions. The enzyme reaction was stopped with 0.1 ml of 1M sodium hydroxide. The p-nitrophenol formed was measured at 405 nm. Aminopeptidase N (APN) activity was also determined by spectrophotometrically, using p-nitroanilide as a substrate.[12] Enzyme activities were determined in unstimulated, concanavalin A (Con A)-stimulated and pokeweed mitogen (PWM)-stimulated lymphocytes.

Statistical Analysis

Data were analyzed using statistical software Jandel SigmaStat® for Windows Version 2.0. Student`s t-test and non-parametric Mann-Whitney Rank Sum Test were used when appropriate, and data were expressed as means ± SD or medians with range in parentheses. Parameters were correlated using simple linear regression test. A p value of less than 0.05 was considered statistically significant.

RESULTS

Baseline Characteristics

Baseline Anthropometric And biochemical characteristics for all examined groups are given in Table 1. Patients with membranous nephropathy, IgA, lupus, and diabetic nephropathy all had a decreased creatinine clearance and increased creatinine compared to the control group. They had also a significantly decreased hemoglobin level. Total protein was decreased in patients with membranous, diabetic, and lupus nephropathy. However, serum albumin was significantly decreased only in membranous and diabetic nephropathy. Diabetic patients had a high glycemia. Triglycerides were significantly increased in patients with membranous, IgA, and diabetic nephropathy. A significant inflammation was demonstrated by an increased CRP in all groups compared to control, as well as an increased fibrinogen in lupus and diabetic nephropathy.

Table 1 Baseline anthropometric and biochemical characteristics

	Membranous nephropathy	IgA nephropathy	Lupus nephropathy	Diabetic nephropathy	Control group
N (M:F)	10 (6:4)	38 (24:14)	29 (4:25)	51 (28:23)	50 (25:25)
Age (years)	59.6 ± 7.4^†	51.6 ± 12.2	43.0 ± 10.5	56.4 ± 12.5^†	47.7 ± 11.6
Hgb (g/dl)	12.33 ± 2.07^†	12.81 ± 2.20^‡	11.13 ± 1.65	11.16 ± 1.95^‡	13.99 ± 1.06
WBC (×10^9/mL)	6.81 ± 2.11	6.81 ± 2.02	5.15 ± 1.66^‡	7.39 ± 2.08^‡	5.97 ± 1.34
Creatinine (μmol/L)	122.93 ± 92.57^‡	202.63 ± 184.21	107.45 ± 64.51^†	319.37 ± 295.36	74.04 ± 11.47
CCr (mL/min)	70.33 ± 31.27^†	66.25 ± 37.39	75.30 ± 21.58	33.86 ± 16.72	109.90 ± 16.41
TP (g/L)	59.28±10.32	70.17 ± 8.80	68.95 ± 6.53^†	67.36 ± 7.91^†	74.43 ± 4.88
Albumin (g/L)	34.70 ± 8.30^†	41.56 ± 5.02	40.55 ± 3.82	39.80 ± 5.38^‡	41.85 ± 3.87
TC (mmol/L)	8.23 ± 3.07	6.72 ± 1.96	5.45 ± 1.27	5.62 ± 1.39	5.87 ± 0.96
TG (mmol/L)	2.61 ± 1.09^‡	2.85 ± 1.88^†	1.97 ± 1.19	2.25 ± 1.02^†	1.67 ± 1.12
Glycemia (mmol/L)	5.09 ± 0.57^‡	4.93 ± 0.58	4.33 ± 0.69	10.48 ± 4.32	5.48 ± 0.45
CRP (mg/dL)	5.43 ± 1.07	3.18 ± 2.03	4.27 ± 3.35	5.16 ± 3.68	1.43 ± 1.01
Fibrinogen (g/L)	4.77 ± 2.61	4.24 ± 1.09	5.13 ± 1.89^‡	5.15 ± 2.51^‡	3.91 ± 1.22

Results are given as means ± SD.

*p < 0.001 vs. control group.

^†p < 0.01 vs. control group.

^‡p < 0.05 vs. control group.

Abbreviations: N (M:F) = number (male:female), Hgb = hemoglobin, WBC = white blood cells, CCr = creatinine clearence, TP = total proteins, TC = total cholesterol, TG = triglycerides, CRP = C reactive protein.

Urinary and Serum Enzyme Activities

Serum PC-1 and APN activities were significantly higher than the control level in membranous nephropathy group, as well as serum PC-1 and DPP IV activities in the IgA nephropathy group (p < 0.05; see Table 2).

Table 2 Serum and urinary enzyme activities

	Membranous nephropathy	IgA nephropathy	Lupus nephropathy	Diabetic nephropathy	Control group
PC-1
Serum (U/L)	4.75 ± 1.09^†	4.60 ± 1.06^†	3.86 ± 0.78	3.61 ± 1.05	3.94 ± 0.68
Urine (U/mmol creatinine)	0.02 (0.002–0.212)	0.04^B (0.003–0.154)	0.01 (0.002–0.217)	0.018 (0.001–0.007)	0.01 (0.001–0.084)
APN
Serum (U/L)	25.58 ± 4.38^†	22.09 ± 5.72	20.55 ± 3.83	20.23 ± 8.21	21.73 ± 4.14
Urine (U/mmol creatinine)	0.14 (0.05–0.71)	0.07 (0.04–0.28)	0.09 (0.05–0.41)	0.20 (0.05–1.40)	0.09 (0.04–0.33)
NAGA
Serum (U/L)	NA	NA	NA	NA	NA
Urine (U/mmol creatinine)	1.14 (0.31–3.80)	0.58 (0.02–2.14)	0.40 (0.10–1.83)	0.47 (0.03–2.29)	0.34 (0.18–1.10)
DPP IV
Serum (U/L)	17.26 ± 6.15	18.97 ± 4.60^†	NA	14.29 ± 4.00	16.17 ± 2.25
Urine (U/mmol creatinine)	0.20 (0.05–0.32)	0.17 (0.05–0.65)	NA	0.31 (0.05–1.64)	0.18 (0.05–0.63)

Results for serum enzyme activities are given as means ± SD, while results for urinary enzyme activities are given as medians with range in parentheses.

*p < 0.01 vs. control group.

^†p < 0.05 vs. control group.

Abbreviations: PC-1 = plasma cell glycoprotein 1, APN = aminopeptidase N, NAGA = N-acetyl-β-D-glucosaminidase, DPP IV = dipeptidylpeptidase IV, NA = not applicable.

Urinary PC-1 excretion in IgA nephropathy group was significantly higher (p < 0.05) than in the control group (see Table 2). Urinary NAGA excretion was markedly (p < 0.01) higher in membranous nephropathy group, compared to healthy controls. APN excretion in diabetic nephropathy group was significantly higher (p < 0.01) than in healthy controls.

Lymphocyte Ectoenzyme Activities

No significant between-group differences were found in PC-1 and APN activities in unstimulated, Con-A, and PWM-stimulated lymphocytes (see Table 3).

Table 3 Lymphocyte ectoenzyme activities

	Membranous nephropathy	IgA nephropathy	Lupus nephropathy	Controlgroup
PC-1 (nmol/min/10^6 ly)
Unstimulated	12.70 (10.30–20.30)	11.95 (9.85–28.00)	11.45 (8.44–19.30)	11.60 (9.38–18.00)
ConA-stimulated	12.30 (10.20–20.10)	11.95 (9.65–17.90)	12.60 (7.57–18.30)	12.70 (9.12–17.50)
PWM-stimulated	12.10 (9.98–18.30)	12.20 (8.64–18.70)	10.95 (8.50–16.50)	12.00 (8.98–18.20)
APN (nmol/min/10^6 ly)
Unstimulated	72.15 (42.30–93.30)	66.20 (39.50–117.70)	71.30 (45.80–160.00)	70.40 (42.60–105.30)
ConA-stimulated	49.75 (34.10–62.30)	50.70 (37.70–65.80)	57.90 (30.50–114.20)	54.80 (39.30–82.80)
PWM-stimulated	40.15 (27.30–51.50)	37.45 (28.90–46.80)	40.40 (27.30–60.00)	39.80 (26.50–55.60)

Results are given as medians with range in parentheses.

Abbreviations: PC-1 = plasma cell glycoprotein 1, APN = aminopeptidase N, ConA = concanavalin A, PWM = pokeweed mitogen.

Correlation of Enzymes with Proteinuria and Glomerular Filtration Rate

Serum and urine enzyme activities were correlated with 24h proteinuria and creatinine clearance (see Table 4). Significant correlation (p < 0.05) in membranous nephropathy of 24h proteinuria with serum PC-1 and urine NAGA was obtained; CCr correlated significantly with urinary NAGA. In IgA nephropathy, 24h proteinuria correlated with urinary NAGA; CCr correlated significantly with urinary APN and DPP IV. In diabetic nephropathy, CCr correlated significantly with urinary PC-1. Significant correlation in lupus nephropathy of 24h proteinuria with urinary NAGA and PC-1 was obtained; CCr correlated significantly with serum APN and urine NAGA. Additionally, in lupus nephropathy, plasma C₃ complement component was found to correlate with serum APN and Con-A- and PWM-stimulated APN activity.

Table 4 Correlation of serum and urine enzyme activities with proteinuria and creatinine clearance

	P	R
Membranous nephropathy
Prot. 24h, PC-1 serum	< 0.01	0.77
Prot. 24h, NAGA urine	< 0.01	0.82
CCr – DPP IV urine	< 0.05	0.71
IgA nephropathy
Prot. 24h, NAGA urine	< 0.01	0.41
CCr, APN urine	< 0.01	0.75
CCr, DPP IV urine	< 0.01	0.71
Lupus nephritis
Prot. 24h, NAGA urine	< 0.001	0.67
Prot. 24h, PC-1 urine	< 0.01	0.55
CCr, APN serum	< 0.05	0.65
CCr, NAGA urine	< 0.05	0.43
CCr, APN (ConA-stimulated Ly)	< 0.05	0.64
CCr, APN ((PWM-stimulated Ly)	< 0.05	0.54
C3, APN (ConA-stimulated Ly)	< 0.05	0.55
C3, APN (PWM-stimulated Ly)	< 0.05	0.59
C3, APN serum	< 0.05	0.63
Diabetic nephropathy
CCr, PC-1 urine	< 0.01	0.39

Abbreviations: PC-1 = plasma cell glycoprotein 1, APN = aminopeptidase N, NAGA = N-acetyl-β-D-glucosaminidase, DPP IV = dipeptidylpeptidase IV, ConA = concanavalin A, PWM = pokeweed mitogen, Prot. 24h = 24h proteinuria, CCr = creatinine clearance.

Diabetic Nephropathy

Anthropometric and biochemical characteristics in diabetic nephropathy subgroups (patients with type 1 and type 2 diabetes mellitus, including those with micro- or macroalbuminuria) are given in Table 5. DM type 1 patients with microalbuminuria were significantly younger than DM type 2 patients. DM type 2 patients with macroalbuminuria had significantly increased serum creatinine and decreased creatinine clearance compared to the two other groups. Glycemia was not adequately controlled, especially in type 1 DM patients.

Table 5 Anthropometric and biochemical characteristics in patients with type 1 DM and type 2 DM with microalbuminuria and macroalbuminuria

	DM type 1 with microalbuminuria (n = 9)	DM type 2 with microalbuminuria (n = 11)	DM type 2 with macroalbuminuria (n = 31)
N (M:F)	4:5	10:1	14:17
Age (years)	40.1 ± 13.8^‡	57.6 ± 10.3	60.6 ± 8.6
Hgb (g/dL)	13.53 ± 2.34^§	14.49 ± 1.02^‡	11.72 ± 1.99
WBC (×10^9/mL)	5.66 ± 1.76	6.65 ± 1.50	7.19 ± 2.09
Creatinine (μmol/L)	81.56 ± 17.11^‡	92.82 ± 19.09^‡	214.1 ± 174.1
CCr (mL/min)	93.56 ± 23.04^‡	111.11 ± 40.09^‡	43.50 ± 24.01
TP (g/L)	72.87 ± 12.11	74.92 ± 3.99	70.74 ± 8.22
Albumin (g/L)	39.20 ± 8.13	41.04 ± 2.92	39.54 ± 5.19
TC (mmol/L)	4.95 ± 1.01^†	5.88 ± 0.82	5.73 ± 1.59
TG (mmol/L)	1.69 ± 0.82	2.89 ± 0.85^││	2.19 ± 1.03
Glycemia (mmol/L)	15.16 ± 6.15^§	10.11 ± 1.73	9.25 ± 3.44
CRP (mg/dL)	4.77 ± 0.97	4.66 ± 1.69	5.47 ± 4.66
Fibrinogen (g/L)	3.72 ± 0.77	4.59 ± 1.22	5.76 ± 2.96

Results are given as means ± SD.

*p < 0.01 vs. DM type 2 with microalbuminuria.

^†p < 0.05 vs. DM type 2 with microalbuminuria.

^‡p < 0.001 vs. DM type 2 with macroalbuminuria.

^§p < 0.01 vs. DM type 2 with macroalbuminuria.

^││p < 0.05 vs. DM type 2 with macroalbuminuria.

Abbreviations: N (M:F) = number (male:female), Hgb = hemoglobin, WBC = white blood cells, CCr = creatinine clearance; TP = total protein, TC = total cholesterol, TG = triglycerides, CRP = C reactive protein.

Urinary APN activity was significantly (p < 0.01) higher in both type 1 and type 2 diabetic patients with microalbuminuria, as well as urinary NAGA and DPP IV activities in type 2 diabetics with microalbuminuria (p < 0.01 and p < 0.05, respectively) compared to controls (see Table 6). However, significantly lower serum DPP IV activity was observed in type 2 diabetics with microalbuminuria, as well as significantly lower serum APN activity in type 2 diabetics with macroalbuminuria, compared to controls (p < 0.05).

Table 6 Serum and urinary enzyme activities in patients with type 1 DM and type 2 DM with microalbuminuria and macroalbuminuria

	DM type 1 (n = 9)	DM type 2 with microalbuminuria (n = 11)	DM type 2 with macroalbuminuria (n = 31)	Control group (n = 50)
PC-1
Serum (U/L)	3.38 ± 0.80	3.65 ± 1.18	3.37 ± 1.04	3.94 ± 0.68
Urine (U/mmol Cr)	0.018 (0.012–0.050)	0.03 (0.001–0.080)	0.01 (0.001–0.039)	0.01 (0.001–0.084)
APN
Serum (U/L)	21.00 ± 5.98	18.91 ± 11.84	18.18 ± 6.99^†	22.27 ± 4.14
Urine (U/mmol Cr)	0.30 (0.09–0.51)	0.35 (0.07–1.40)	0.16 (0.05–0.66)	0.09 (0.04–0.33)
NAGA
Serum (U/L)	NA	NA	NA	NA
Urine (U/mmol Cr)	0.42 (0.13–0.92)	0.72 (0.06–2.29)	0.42 (0.03–0.71)	0.34 (0.18–1.10)
DPP IV
Serum (U/L)	12.78 ± 4.12^†	12.18 ± 6.22^†	15.16 ± 2.96	16.39 ± 2.25
Urine (U/mmol Cr)	0.42 (0.13–0.75)	0.50 (0.07–1.64)^†	0.21 (0.05–0.66)	0.18 (0.05–0.63)

Results for serum enzyme activities are given as means ± SD, while results for urinary enzyme activities are given as medians with range in parentheses.

*p < 0.01 vs. control group.

^†p < 0.05 vs. control group.

DISCUSSION

Previous studies have suggested that proteinuria resulting from glomerular disease has a direct role in activating the cascade initiated by epithelial cell injury. High absorption rates of proteins may lead to striking changes in tubular morphology, including dramatic enlargement of protein absorption droplets and loss of brush border structure, suggesting pathologic injury.[21]

A recent study demonstrated the highest increase urinary NAGA activity in patients with primary glomerulonephritis.[9] In the present study, in membranous nephropathy, urinary NAGA excretion was significantly increased compared to that of controls. Furthermore, this study also has shown a significant correlation between proteinuria and urinary NAGA excretion in patients with membranous nephropathy. It is important to emphasize that in the majority of our patients, the disease was manifested by nephrotic range of proteinuria. This data suggests that urinary NAGA activity may be indicative of tubular damage with lysosomal cell injury. Increased serum PC-1 and APN activity in these patients may be the consequence of the damage of brush border of the proximal tubule. Because no correlation between these findings and decline in renal function was observed, these might be early markers of tubular dysfunction, prior to interstitial fibrosis, and could have important role in making therapeutic decision. Contrary to previous data, a significant correlation between the decline of renal function and urinary DPP IV activity was observed. Increased urinary excretion of DPP IV, an intrinsic membrane glycoprotein localized on the proximal tubule brush border, and glomerular visceral epithelial cells may represent adverse glomerular cells damage. This observation is confirmed by a similar correlation between creatinine clearance decline and urinary DPP IV activity in patients with IgA nephropathy.

DPP IV could be the therapeutic target in type 2 diabetes mellitus.[17] The major clinical trials with DPP-IV inhibitors as monotherapy and as add-on therapy in patients with type 2 diabetes were reviewed.[22] The magnitude of HbA1c reduction with DPP-IV inhibitors depends upon the pretreatment HbA1c values, but there seems to be no change in body weight and very low rates of hypoglycemia and gastrointestinal disturbance with these agents.

The therapeutic modification of the PC-1 expression was demonstrated in insulin resistant type 2 diabetics after a three-month metformin treatment.[23] The study has shown an increased activity of lymphocyte PC-1 in Type 2 diabetes and its reversal by three-month metformin treatment, corresponding to the improvement of insulin sensitivity. Data obtained are consistent with a role of PC-1 in insulin resistance and suggest a new mechanism of action for metformin via PC-1 inhibition.

The predictive value of NAGA excretion on functional outcome and response to therapy was demonstrated.[24] Using the Cox model, in 74 patients with idiopathic membranous nephropathy (IMN), only NAGA excretion, and not 24-h proteinuria, predicted progression to chronic renal failure (CRF). In 18 patients with minimal change disease (MCD), NAGA excretion values were below the chosen cutoff, and 90% of them developed remission. Response to immunosuppressive therapy was significantly different in patients with NAGA excretion values below or above the cutoff. Urinary NAGA excretion can be considered as a reliable marker of the tubulotoxicity of proteinuria in the early stage of IMN, FSGS, and MCD. Its determination may be a non-invasive, useful test for the early identification of patients who will subsequently develop CRF or clinical remission and responsiveness to therapy. Urinary NAGA excretion is elevated in patients with type 2 DM compared with healthy individuals and increases as nephropathy progresses.[25] Although urinary NAGA excretion rate was shown as a less sensitive staging parameter, being significantly increased when compared to control group only in macroalbuminuria and CRF groups, it significantly correlated with 99mTc-DTPA clearance rate and 99mTc-DMSA tubular fixation.[25] Urinary NAGA proved comparable to urinary albumin excretion when analyzed with respect to preexistence and the development of severe macrovascular disease.[26] It could be used as a marker for efficacy of treatment. Pentoxifylline (PTF) administration, 1200 mg/d for 4 months to 30 patients, was effective in reducing proteinuria and urinary NAG excretion in these patients. The findings suggest that PTF may have beneficial effects on tubulointerstitial damage in diabetic kidney disease.[27]

Urinary NAGA was found to be increased in children with insulin-dependent diabetes mellitus (IDDM) without any clinical evidence of nephropathy.[28] Such increased excretion correlated with the plasma HbAlc level. There is a tubular dysfunction in the early stage of IDDM children even before there is any clinical evidence of nephropathy, and urinary NAGA may reflect glycemic control in such patients.

An increased serum PC-1 activity, as well as urinary PC-1 activity in patients with IgA nephropathy, independent of renal function or proteinuria as markers of renal damage, was observed in this study. These findings may suggest possible, unknown mechanism by which stimulated PC-1 activity leads to glomerular and tubular injury.

A significant correlation between decline of glomerular filtration rate, measured by creatinine clearance, and increased urinary excretion of APN was observed in this study. The data could suggest that increased urinary APN activity represent severe renal injury and adverse outcome. Compared to healthy controls, urinary APN and NAGA were found significantly elevated in glomerulonephritis patients.[28] APN indicates very early tubular impairment and, in some cases, APN is elevated although NAGA is still within normal ranges.[13]

In conclusion, damage of tubules in primary glomerulonephritis, lupus nephritis, and diabetic nephropathy is accompanied by a release of several tubular enzymes, with possible diagnostic and prognostic significance. Increased serum PC-1, APN, and DPP IV activities could be also of diagnostic significance.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ACKNOWLEDGMENTS

This work was supported by grant number 145004 from the Ministry of Science of Serbia. None of the authors has any financial interest to disclose.