Renal Enzymes and Microglobulins in Patients with Rheumathoid Arthritis

The urine enzymes usually originate from epithelium cells and glands of urogenital system, plasma, leucocytes, erythrocytes and kidneys [1]. Approximately 40 [2-4] different enzymes from different groups appear in urine: transferases, lyases, oxidoreductases, different isomerases and ligases which usually are not found in urine. The appearance of such great number of enzymes in urine is due to the kidney’s dominant function excretion. Brushed epithelium of proximal tubules is the location for alanine aminopeptidase (AAP) 90%, alkaline phosphatase (AP) -70% and γ -glutamyl transferase (γGT) 50% from the total activity of these kidney enzymes [5]. Brushed epithelium is very sensitive even in physiological conditions, thus definite dispensation of superficial enzymes can be used as a marker for renal damage both primary and secondary, caused by drugs or toxins [6]. Tubular lysosomal system is very dynamic system and low level of lysosomal enzymes found in normal urine is a result of normal exocytic and pinocytic activity of tubular epithelial cells [7]. The extent of enzymuria depends on location and damage intensity. Increased enzymatic activity is a reflection of disease activity and kidney’s residual functional capacity [8]. Renal tubular damage initially affects lysosomal /plazma cell membrane system, causing enzymatic loss in urine in early phase. Further increase in enzymatic excretion is connected with cell structural damage causing cell necrosis. Elimination of toxic stimulus is followed by reduction of urine enzymatic activity and tubular regeneration. The aim of this study is to define the effect of untreated rheumatoid arthritis on tubular function and sensitivity of brush border epithelium of renal proximal tubules. AAP, γ -GT and β2microglobulin (β2M) in urine are used as indicators for proximal tubular damage.


Introduction
The urine enzymes usually originate from epithelium cells and glands of urogenital system, plasma, leucocytes, erythrocytes and kidneys [1]. Approximately 40 [2][3][4] different enzymes from different groups appear in urine: transferases, lyases, oxidoreductases, different isomerases and ligases which usually are not found in urine. The appearance of such great number of enzymes in urine is due to the kidney's dominant function -excretion. Brushed epithelium of proximal tubules is the location for alanine aminopeptidase (AAP) -90%, alkaline phosphatase (AP) -70% and γ -glutamyl transferase (γ-GT) -50% from the total activity of these kidney enzymes [5]. Brushed epithelium is very sensitive even in physiological conditions, thus definite dispensation of superficial enzymes can be used as a marker for renal damage both primary and secondary, caused by drugs or toxins [6]. Tubular lysosomal system is very dynamic system and low level of lysosomal enzymes found in normal urine is a result of normal exocytic and pinocytic activity of tubular epithelial cells [7]. The extent of enzymuria depends on location and damage intensity. Increased enzymatic activity is a reflection of disease activity and kidney's residual functional capacity [8]. Renal tubular damage initially affects lysosomal /plazma cell membrane system, causing enzymatic loss in urine in early phase. Further increase in enzymatic excretion is connected with cell structural damage causing cell necrosis. Elimination of toxic stimulus is followed by reduction of urine enzymatic activity and tubular regeneration. The aim of this study is to define the effect of untreated rheumatoid arthritis on tubular function and sensitivity of brush border epithelium of renal proximal tubules. AAP, γ -GT and β2-microglobulin (β2M) in urine are used as indicators for proximal tubular damage.
2. Intermediate proteins normally filtered in glomerulus in very small quantity, while the rest is reabsorbed in tubules (microalbumin, transferin).
Alanine aminopeptidase (AAP) arylamidase amino acid, amino peptidase, α-aminoacyl-peptidyl hydrolase (microsomal EC 3.4.11.2, earlier 3.4.1.2) is hydrolytic product of peptides, amides and p-nitroanilide. AAP is found in many tissues such as kidneys, intestine, lung and liver. AAP in different tissues has different electrophoretic conductivity. This enzyme has at least five [5] different isoenzymes, distinguished by immunological and electrophoretic features and ion change chromatography [15]. Normal serum contains only one isoenzyme, while in liver, biliary or pancreatic disease additional fractions are found. The enzyme is found in urine [16].
glutathione. High concentrations of enzymes are found in kidneys (renal proximal tubules), pancreas (acinar cells), prostate and liver. γ-GT is located mainly in external parts of plasma membrane. Isoenzymes of γ-GT in serum are consequence of different posttranslational modifications such as modification of carbohydrate part of molecule of γ-GT, formation of complexes with lipoproteins [17,18]. Due to the differences in carbohydrate parts of the γ-GT molecule the isoenzymes are found in different tissues (liver, pancreas, kidney and duodenum). Although the peptide part of enzyme is the same in the tissue that originate, these isoenzymes differ in kinetic, electrophoretic and immunologic features [19]. Renal tubular function could be evaluated by measuring the excretion of low molecular proteins in urine.
β 2-microglobulin (β2M) in urine is used for detection of tubular malfunction in glomerulonephritis [20] and is often used as sensitive marker for evaluation of renal function [21][22][23][24][25]. β2M is a polypeptide with small molecular weight (11.815 daltons). It contains light chain of main histocompatibility antigen (HLA) and influences the production of rheumatoid factor (IgM class) [26]. β2M is found in serum and urine in healthy individuals [27]. 95% of free β2M is ultra-filtrated in renal glomerulus and almost complete (99.99%) is reabsorbed via proximal tubular endocytosis and finally is catabolized in amino acid in healthy individuals. Usually it is detected in traces in urine. Disturbance in glomerular filtration leads to increase in serum β2M, while tubular damage leads to increase in urine β2M. Serum β2M concentration is dependent on GFR and shows significant negative correlation with inulin clearance. Serum β2M level could serve as an index of damage intensity of renal glomeruli. In pathological conditions increased amount of β2M are excreted in urine. This happens when serum β2M concentration exceeds renal threshold. Serum level of β2M is dependent on synthesis or excretion in serum and is in relation with clearance. This happens in patients with inflammatory diseases such as rheumathoid arthritis [28], SLE [29], Sjogren's syndrome, Crohn's disease [30], cancer and liver damage. Urine β2M concentration can be increased in conditions when reabsorption is decreased due to renal proximal tubule damage. It results in urine β2M increased concentration and enables distinction between renal proximal tubular and glomerular damage.
β2M is used for GFR and for renal tubular function evaluation, especially for toxic tubular damage caused by heavy metals (cadmium and lead) and as screening test for early detection of Balkan's nephritis in endemic regions. β2M is unstable in urine pH<6 and it is recommended the urine to be alkalized with bicarbonates before it is processed. β2M is considered the earliest protein in tubular proteinuria. It suggests an asymptomatic renal dysfunction in RA patients.

Material and Methods
Diagnosis of rheumatoid arthritis (RA) was based of the revised diagnostic criteria for classification of RA, suggested in 1987 by the American Association for Rheumatism (ARA) [31]. Patient has to fulfill 4 out of 7 ACR criteria in order to be included in the group with RA. Criteria from one 1 to four 4 have to be present at least 6 weeks. 70 participants were included in the study: 35 RA patients (28 female, 7 male) and 35 participants (18 female, 17 male) as healthy control group. Their average age in the group with RA was 56-68 years (±6,79) (range 40-65 years), but in healthy control group 46,2 years (±12,49) (range 29-65 years). The average period from the beginning of disease was 43, 97 months (±45,23) (in interval 1-168 ). All the participants denied past or present renal disease. Three patients were previously treated with oral steroids, while nobody used NSAIL. The rest of the patients denied drug use before inclusion in the study.

Inclusion criteria
newly diagnosed patients with RA, not treated, age 18-65 years.
2. Patients with previous history of renal, hematologic, cardiovascular, neurologic, liver and lung damage, diseases of the spleen and thyroid gland.
3. Patients who take drugs from basic line.

Patients treated with antibiotics and salicylates in period <6
months from the beginning of the study.
6. Patients treated with antihypertensive, diabetic and cardiac therapy.
7. Hypersensitive to some drugs or their component.

Patients with previous history for blood transfusion and overweight.
9. Patients with glycemia in 0 spot or increased level of degraded products: creatinine in serum and urine, hematuria/ proteinuria, urea in serum and disorder of hematologic and enzymatic status.
All patients took part voluntarily in the study, so the ethical criteria were fulfilled.

Clinical evaluation of disease activity
The disease activity was evaluated by subspecialist using DAS 28 index (Disease Activity Score -DAS 28) [32][33][34][35]. The index is mathematical formula, so we can get uniquely composed quantitative score, which consists of palpation of painful sensitive joints (max number 28), swollen joints (max number 28), ESR and patient's global assessment of disease activity (0-100mm Visual Analogous Scale -VA ) and the morning stiffness. DAS 28 index is ranked from 0 to 10 and score <3.2 ranks the disease as low active. The assessment of glomerular filtration rate (GFR) is calculated with Cockroft & Gault's formula. [36]
ESR was determined using Westergren's quantitative method for ESR.
Reference values: 7-8mm for male, 11-16mm for female. γ-glutamil transpeptidase (γ-GT) is detected with IFCC method [42]. The methods of measuring the activity of this enzyme in serum use aromatic amides as substratum (γ-glutamylanilide and γ-glutamylnaftilamide). The most often used artificial substratum peptide analog is γ-glutamyl-p-nitroanilide, offering possibility for kinetic and colorimetric determination of the enzymatic activity [43,44]. γ-glutamyl-p-nitroanilide later is changed with L-γ-3karboksi-4-nitroanalid (Glukan), because of higher dissolution [45]. As acceptor of the substratum and puffer is used glycin-glycin, getting higher catalytic activity. This method is standardized by International Federation for Clinical Chemistry (IFCC) and is considered as reference method. IFCC method for measurement of the concentration of catalytic activity of γ-GT in serum and urine is based on the principles developed by Orlowski and Meiser [43] and Szasz [42]. This method has modifications developed by Persijin and Van der Slik [44]. L-γ-glutamyl-3-carboxi -4-nitroanilide is used as a donor of the substratum. In IFCC method Tris (hydroxymethyl) aminoethane is changed with glycin-glycin, used as a buffer and acceptor of the substratum. Magnesium (Mg) earlier used for keeping L-γ-glutamyl-4-nitroanilide in solution in IFCC method is omitted.

Detection of β2-microglobulin (β2M) in urine by MEIA method (Microparticle Enzyme Immunoaassay) (Abbot A x sym system)
A x sim β2M detection is based on MEIA technology (Microparticle Enzyme immunoassay), used for quantitative detection of β2M in serum, plasma and urine in patients with RA and renal dysfunction. The reaction consists of interaction of β2M with anti-β2M antibody, forming an interrelated complex. The complex reacts with Matrix cell and is tightly connected to them. A conjugate of alkaline phosphatase is added, which connects with the complex forming sandwich-complex. 4-Methylumbelliferyl Phosphate (4-MUP) is added on this complex; it reacts with alkaline phosphatase from the complex and fluorescent product Methylumbelliferon with light blue color is obtained. The degree of optical fluorescence directly proportionally determines β2M concentration. It is determined automatically (Abbot A x sym system). β2M is very sensitive to pH changes in urine, because it decomposes rapidly in low pH values (<pH 6.0). So, if the urine is acid, it has to be alkalized.

Statistical Analysis
The difference between two arithmetic means was tested with Student's "t-test", comparing the middle values of the certain numerical parameters between two groups. Wilcoxon-matched test was used for independent samples. Sensitivity and prediction for positive and negative test of examined values were defined with the test of sensitivity and specificity. P value under 0.05 was taken as statistically significant. Data processing was done with statistical package -Statistica 7.0. patients (48.57%) were RF positive. Among 18 RF negative patients, AAP enzymuria was present in 14 patients (77.77%), γ-GT was present in 10 patients (55.55%), while β2M in urine was not present at all (0%). Among 35 RA patients sensitivity of AAP was 68.57%, sensitivity of γ-GT was 45.71%, sensitivity of β2M was 0%, while sensitivity of RF was 48.57%. Among 17 RF positive patients, the presence of γ-GT was detected in 6 patients (35.29%), the presence of AAP was in 10 patients (58.82%), while the presence of β2M was not detected in urine (0%). Among healthy control group 7 patients (20%) were AAP positive, 6 patients (17.14%) γ-GT positive and 1 patient (2.85%) showed presence of β2M in urine. RF appeared in 2 patients (5.71%) ( Table 1).

Diagnostic value of alanine aminopeptidase (microsomal AAP), γ-GT and β2M in urine in RA patients
For AAP, γ-GT and β 2M and for other laboratory variables in RA, sensitivity, specificity, predictable value for positive and negative test are show in (Table 2). AAP had better diagnostic performances than -GT and β2M in terms of sensitivity (sensitivity 68.57% vs 45.71% vs 0%) and specificity (specificity 80% vs 82.85% vs 97,14%) in detection of renal tubular damage in untreated RA.   1. RF negative patients had higher value of AAP than RF positive patients (Table 1).

5.
There was not statistical correlation between γ-GT in RA patients and control healthy group (p= 0.308160), β2M in RA and healthy control group (p= 0.05).

Discussion
Elevation in urine enzymes' activity could indicate primary renal tubular damage, because of their location in brush border area, such as microsomal AAP (E.C 3.4.11.2) and tubular lysozyme (NAG E.C.3.2.1.30). They could be used in early diagnosis of acute renal failure caused by immunosuppressive drugs, contrast materials, antibiotics and cadmium exposition [46][47][48][49][50][51][52]. Urine enzymatic activity normally is low and is increased in case of the renal tubular damage [53]. Urine enzymes, especially NAG, AAP, AP are very sensitive indicators of renal parenchymal damage in comparison with functional measurements such as glomerular filtration rate (GFR), creatinine and inulin clearance. Relatively low sensitivity of GFR can be explained with large functional kidney and its great compensatory ability [53]. Sensitivity of AAP is higher compared with γ-GT and β 2M (68,57% vs 45,71% vs 0%) with approximately equal specificity (80% vs 82,85% vs 97,14%). The other standard routine analyses used for evaluation of renal function showed low sensitivity (creatinine in serum and urine, serum urea (8.57% vs 25.71% vs 11.42%). RF negativity had influence on the presence of AAP enzymuria. It was presented in RF negative patients with DAS 28>3.2 with much higher β2M than in RF negative patients with DAS 28>3.2 (0.07 ± 0.04) (0.01-0.15) vs (0.047 ± 0.039) (0.01-0.13), but that was not the case with the intensity of γ-GT, where RF positivity had different meaning. The duration of the disease in months and AAP, γ-GT and β2M enzymuria (p=0.00) showed that untreated RA had an influence on renal tissue as one of visceral manifestation of the disease. Enzymes originating from primary damaged proximal tubular brush border area in untreated RA had higher sensitivity.

Conclusion
AAP has higher sensitivity then γ-GT and β2M in detection of asymptomatic renal damage in untreated RA. AAP and γ-GT can be used in everyday clinical practice in diagnose of early asymptomatic renal damage.