Production of M2000 (β-d -mannuronic acid) and its therapeutic effect on experimental nephritis

Environmental Toxicology and Pharmacology 24 (2007) 60–66

Production of M2000 (␤-d-mannuronic acid) and its therapeutic effect on experimental nephritis A. Mirshafiey a,∗ , B. Rehm b , R. Safari Abhari a , Z. Borzooy a , M. Sotoude a , A. Razavi a a

Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Box 6446, 14155 Tehran, Iran b Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand Received 27 January 2007; accepted 11 February 2007 Available online 16 February 2007

Abstract The present research introduces the method of Production of M2000 (␤-d-mannuronic acid) and its therapeutic effect on experimental model of nephritis. M2000 was produced using enzymatic and chemical procedure on prepared alginate from Pseudomonas fluorescens. The experimental glomerulonephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of bovine serum albumin (BSA). M2000 solution (30 mg/kg) was administered intraperitoneally at regular 48-h intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, BUN, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. PMN infiltration and glomerular immune complex deposition was less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200 ␮g/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug. Conclusions: In this research, for the first time we introduced the procedure of production of M2000 (␤-d-mannuronic acid) and our data suggest that treatment with M2000, as a novel anti-inflammatory drug can reduce proteinuria, diminish antibody production and suppress the progression of disease in experimental model of glomerulonephritis. © 2007 Published by Elsevier B.V. Keywords: Glomerulonephritis; Anti-inflammatory; NSAIDs; Proteinuria; M2000

1. Introduction Alginates are natural copolymers comprised of ␤-dmannuronate (M-block) and ␣-l-guluronate (G-block) linked by 1 → 4 glycosidic linkage. They are synthesized by bacteria belonging to the genera Pseudomonas and Azotobacter and brown sea-weeds, and the M-blocks of the bacteria, but not seaweed polymers, are to a variable extent acetylated at positions

∗

Corresponding author. Fax: +98 21 66462267. E-mail address: a [email protected] (A. Mirshafiey).

1382-6689/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.etap.2007.02.002

0–2 and/or 0–3 (Skjak-Braek et al., 1986; Bucke, 1987; Rehm, 1998). The variability in monomer block structures and acetylation which are associated with the source of alginate, strongly affect the physicochemical and rheological properties of the polymer, and the biological basis for the variability is therefore of both scientific and applied importance (Rehm and Valla, 1997). The renal glomerulus is the specialized structure in the kidney responsible for generating over 150 l of plasma ultrafiltrate per day in humans. Certain characteristics of this structure favor involvement in autoimmune diseases (Quigg, 2004). Glomerulonephritis is an important cause of renal failure thought to be

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caused by autoimmune damage to the kidney. While each type of glomerulonephritis begins with a unique initiating stimulus, subsequent common inflammatory and fibrotic events lead to a final pathway of progressive renal damage (Vinen and Oliveira, 2003). Many forms of glomerulonephritis involve immune complex localization in the kidney. Formation of immune complexes in the glomerulus, either deposited from the circulation or generated in situ, can activate the complement system (Tarzi and Cook, 2003; Rougier and Ronco, 2003). The common inflammatory pathways follow with activation of the coagulation and complement cascades and production of proinflammatory cytokines (Hricik et al., 1998). Activation of complement components leads to the production of anaphylatoxins C3a and C5a, C3b, which covalently associates with immune complexes, and the C5b-9 membrane attack complex, as well as chemotaxis of inflammatory cells (Quigg, 2004). On the other hand, Fcgamma receptors expressed on the surface of leukocytes bind the Fc (constant) portion of IgG. They link immune complex deposition to innate immune responses, including Phagocytosis, cytokines release, formation of reactive oxygen species, and antibody-dependent cytotoxicity (Tarzi and Cook, 2003). The coagulation cascade leads to fibrin deposition. Cellular proliferation of parietal epithelial cells in Bowman’s space together with an influx of inflammatory cells such as macrophages and neutrophils results in acute glomerular crescent formation. Cytokine release leads to activation of the glomerular cells themselves and a change in endogenous cell phenotype results in cell proliferation, overproduction of proteases and oxidants, and laying down of extracellular matrix with subsequent fibrosis, perhaps stimulated by factors such as platelet derived growth factor and transforming growth factor beta (Vinen and Oliveira, 2003). On the other side, it has been reported that expression of matrix metalloproteinases (MMPs) by infiltrating and intrinsic renal cells is increased in inflammatory conditions, and may correlate with disease activity of glomerulonephritis, so that increased MMP-2 and MMP-9 activity contributes to glomerular injury and hypertensive remodeling, as well as the over-expression of MMP-2 in glomerular cells might play a critical role in the development of glomerulosclerosis (Camp et al., 2003; Sanders et al., 2004; Zhang et al., 2004). The current study was undertaken to explore the therapeutic potency of M2000 (␤-dmannuronic acid) molecule, as a novel designed non-steroidal anti-inflammatory drug (Mirshafiey et al., 2004, 2005a,b) in experimental model of immune complex glomerulonephritis.

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2.2. Procedure for M2000 production 2.2.1. Cultivation Production of P. fluorescens alginate was performed in liquid PIA medium (shake flasks) containing bacteriological peptone (20 g/l), NaCl (5 g/l) MgCl2 (1.4 g/l), K2 SO4 (10 g/l), and 20 ml of 87% glycerol/liter or in PM5 medium (fermentors) containing fructose (40 g/l), yeast extract (12 g/l), (NH4 )2 SO4 (0.6 g/l), Na2 HPO4 ·2H2 O (2.0 g/l), NaCl (11.7 g/l), and MgSO4 ·7H2 O (0.3 g/l), and clerol FBA622 (antifoam, 0.5 g/l). The media were supplemented with proteases—Alkalase 2.4L and Neutrase 0.5L from Novo Nordisk (0.15 ml/l each in PIA and 0.25 ml/l each in PM5) in order to reduce extracellular alginate-lyase activity. 2.2.2. Alginate isolation and purification Isolation of deacetylated alginate from culture supernatants was performed by adding an equal volume of isopropanol. The precipitate was collected by centrifugation and washed with both 70 and 96% ethanol. The dried precipitate was fully dissolved in a small volume H2 O and DNase I and RNaseA (10 ␮g/ml of each) were added and incubated at 37 ◦ C for 4 h. The Pronase E (12.5 ␮g/ml) was then added and incubated over night at 37 ◦ C. Using the dialysis tubing, one end was clamped and poured the solution into the tube using a glass funnel (the bag should be 2/3 full, allowing room for expansion) and clamped another end. Then it was dialysed against 2.5 l distilled H2 O in a 4–5 l beaker, stirred very slowly using a magnetic stirrer. The water was changed after 2–3 days. The alginate solution was then lyophilized. 2.2.3. Deacetylation Lyophilized alginate was dissolved in water to obtain a 0.5% (w/v) alginate solution and added 0.1 M NaOH and stirred 1 h at RT and neutralized with HCl. Then it was dialysed and lyophilized again. 2.2.4. Hydrolysis Following deacetylating alginate was dissolved in water at concentration 1 mg/ml and Adjusted to pH 2 with 1 M HCl and incubated at 110 ◦ C for 8 h in an autoclave. It was neutralized with 1 M NaOH. 2.2.5. Collection of monomer fraction Gel filtration chromatography (superpeptide column from Amersham Biosciences) was used to remove salt. Uronic acid containing fractions were lyophilized and subjected to anionic exchange chromatography (Rehm, 1998). The monomer fraction was collected and confirmed by TLC. 2.2.6. Uronic acid determination Based on the method of Blumenkrantz and Asboe-Hansen (1973), which is a Galambos’ modified procedure (Galambos, 1967), diphenyl reagent and H2 SO4 Tetraborate Solution was used to build a pink colored chromophor. Using neutral sugars in 10-fold excess give a brown color which interferes with detection of the uronic acids. The absorbance was detected at 520 nm in a spectrometer. There was a linear relationship between the absorbance and the amount of sugar that was present in the original sample. It was non-stoichiometric and therefore it was necessary to prepare a calibration curve using a series of standards of known uronic acid concentrations, pure alginate (Vilim, 1985; Filisetti-Cozzi and Carpita, 1991).

2. Materials and methods 2.3. Animals 2.1. Reagents Pseudomonas fluorescens Pf20118 (request from Prof. S. Valla, Norway). Solution A (0.15% m-hydroxydiphenyl in 0.5% NaOH: dissolving the crystals in 10 ml of fresh 0.5% NaOH and storing the solution in a dark bottle and/or foil wrapped bottle). Solution B (0.0125 M disodiumtetraborate in conc. H2 SO4 solution: preparing uronic acid standards using the stock 1 mg/ml pure alginate provided. Preparing serial dilutions 31.25 ␮g/ml; 62.5 ␮g/ml; 125 ␮g/ml; 250 ␮g/ml). BSA (Fluka, The Netherlands) was dissolved in sterile 0.15 M NaCl solution at 5 and 30 mg/ml concentrations. Complete Freund’s adjuvant (CFA) was obtained from Difco Laboratories, Detroit, MI.

A total of 58 female Sprague–Dawley rats weighing 180 ± 20 g were obtained from the Razi institute (Karaj, Iran) and were housed in our animal facility (temperature, 20–22 ◦ C; humidity, 55–56%; 12-h light/12-h dark cycle; unlimited access to food and water) for at least 1 week before the experiment. Rats were divided randomly into five groups. N: normal group (n = 8); P: patient group (n = 9); T1 and T2: treated patient groups with M2000 and piroxicam, respectively (n = 2 × 8); H: healthy control receiving M2000 (n = 8). Experimental procedures were performed in accordance with the recommendations and Policies of the Iran Pasteur Institute for the protection of animals used for experimental and other scientific purposes.

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2.4. Experimental protocol and treatment Immune complex glomerulonephritis was induced by the method of Yamamoto et al. (1978). Briefly, 8 weeks after subcutaneous immunization with 1 mg of bovine serum albumin (BSA) in complete Freund’s adjuvant, the animals received an intravenous dose of 2 mg BSA daily for 4 weeks. On day 56, therapeutic protocol was developed by intraperitoneally (i.p.) administration of M2000 solution (30 mg/kg) to group T1 and i.p. injection of piroxicam (0.3 mg/kg) to group T2 . A total of 15 i.p. injections, at regular 48-h intervals was considered for M2000 and/or piroxicam. The experiment was terminated on day 84 by killing the rats.

2.5. Measurement of anti-BSA antibody Blood samples obtained from P, T1 and T2 groups in the 6th, 9th and 12th experimental weeks were considered for measurement of anti-BSA antibody. Polyvinylchloride ELISA Plates (Nunc-Immuno Module, Denmark) ere coated with BSA diluted in 0.1 M sodium carbonate buffer pH 9.6 by incubation for 1 h at 37 ◦ C, followed by incubation for 16 h at 4 ◦ C (1 ␮g/well). The wells were washed three times with 0.05% Tween 20 in PBS (PBS-T) and incubated for 1 h at 37 ◦ C with the rat sera diluted 1:1000. Unbound material was removed by three washes with PBS-T. Rabbit anti-rat polyvalent Ig-peroxidase conjugate diluted 1:500 in PBS was incubated at room temperature for 1 h, followed by three washes with PBS-T. The peroxidase was reacted with Ophenylenediamine (Sigma) and H2 O2 (Merck, Germany) for 60 min, and 2N H2 SO4 was added for stopping the reaction. The reaction product was quantified by measuring extinction at 492 nm based on the method of Hogendoorn et al. (1990).

2.6. Assessment of kidney function Measurement of proteinuria was carried out during six stages, from weeks 3 to 12 post-immunization; urines were collected before the intraperitoneal injections of M2000 and/or piroxicam. Urine protein was measured using precipitation by trichloroacetic acid. Creatinine clearance was calculated by measuring creatinine concentration in urine and plasma by the alkaline picrate method (Bousnes and Taussky, 1945). Urine urea and blood urea nitrogen (BUN) was assessed by the oxime method (Evans, 1968).

of 30 mg/kg. In this group, the number and interval of injections were 15 and 48 h, respectively. Three days after the last injection, toxicological studies were done as follows: 1. Assessment of kidney function was carried out based on the measurements of urinary protein excretion, urine and plasma creatinine, BUN and urine urea. 2. Evaluation of serum lipid levels was performed by determining serum triglyceride and cholesterol concentrations. 3. Histological studies renal tissues were assessed using light microscopy. Glomerular lesions were graded on a scale of 0–3 (0, negative; 1, mild; 2, moderate; 3, marked) according to five parameters: hypercellularity, glomerular infiltration of PMN, fibrinoid necrosis, interstitial infiltration, and the presence of tubular casts.

2.10. Cell culture The fibrosarcoma (WEHI-164) cell line was seeded at an initial density of 20,000 cells/well in 96-well tissue culture plate. Cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, penicillin at 100 units/ml, and streptomycin at 100 ␮g/ml, with 5% CO2 , 37 ◦ C and saturated humidity.

2.11. Dose–response analysis Triplicate, two-fold dilutions of M2000, dexamethasone, piroxicam and diclofenac preparations at concentrations of 10–200 ␮g/ml were transferred to overnight cultured cells. Non-treated cells were used as controls. Cells were cultured overnight and then subjected to colorimetric assay. A sample of the media was used for zymography.

2.12. Colorimetric assay After each experiment, cells were washed three times with ice-cold phosphate-buffered saline (PBS), followed by fixation in a 5% formaldehyde solution. Fixed cells were washed three times and stained with 1% crystalviolet. Stained cells were washed, lysed and solublised with 33.3% acetic acid solution. The density of developed purple color was read at 580 nm.

2.7. Evaluation of serum lipid levels 2.13. Zymography Serum concentrations of triglyceride and cholesterol were determined by the routine laboratory tests on the day of sacrifice.

2.8. Histological examinations Kidney specimens were processed using light and immunofluorescence microscopic examination. For light microscopy, renal tissues were fixed by immersion in 10% buffered formalin, embedded in paraffin, and 4-␮m sections were stained with hematoxilin-eosin and periodic acid–Schiff. The severity and extent of glomerular lesions were blindly evaluated in four parameters: hypercellularity; glomerular infiltration of PMN; fibrinoid necrosis and interstitial infiltration. In addition, the existence of tubular casts was considered. These parameters were evaluated by a semi-quantitative method of renal histology using a grading scale of 0–3 (0, negative; 1, mild; 2, moderate; 3, severe). For immunofluorescence microscopy, renal tissues were instantaneously frozen in n-hexane precooled to −70 ◦ C, and 4-␮m cryostat sections were stained with fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG. The degree of deposition of immune complex was graded quantitatively into −, +, 2+, and 3+ on the basis of intensity and distribution.

2.9. Toxicological studies To evaluate the side-effects of M2000 in normal rats, healthy group (H) was considered. This group received only intraperitoneal injections of M2000 at dose

This technique was used for determining gelatinase (collagenase type IV or matrix metalloproteinase type 2, MMP-2) and MMP-9 activity, in conditioned media according to the modified Heussen and Dowdle method (Heussen and Dowdle, 1980). Briefly, aliquots of conditioned media were subjected to electrophoresis in (2 mg/ml) gelatin containing polyacrylamide gels, in the presence of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under non-reducing conditions. The gels underwent electrophoresis for 3 h at a constant voltage of 80 V. After electrophoresis, the gels were washed and gently shaken in three consecutive washings in 2.5% Triton X100 solution to remove SDS. The gel slabs were then incubated at 37 ◦ C overnight in 0.1 M Tris–HCl gelatinase activation buffer (pH 7.4) containing 10 mM CaCl2 and were subsequently stained with 0.5% Coomassie Blue. After intensive destaining, proteolysis areas appeared as clear bands against a blue background. Quantitative evaluation of both surface and intensity of lysis bands, on the basis of grey levels, were compared relative to nontreated control wells and expressed as “Relative Expression” of gelatinolytic activity.

2.14. Statistical analysis Data were expressed as means ± S.D., except for histological scores, which were calculated as means ± S.E.M. Statistical analysis was performed using student’s t-test for parametric data and Mann–Whitney U-test for nonparametric data. P values <0.05 were considered significant.

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3. Results

3.2. Anti-BSA antibody titer

3.1. Effect of M2000 on serum and urine determinants

The changes of the mean levels of anti-BSA antibody titers in the 6th, 9th and 12th experimental weeks are illustrated in Fig. 3. Anti-BSA antibody titers were significantly lower in M2000treated animals (T1 ) than piroxicam (T2 ) and nontreated controls (P) at the end of experiment (day 84).

The changes of the mean levels of urinary protein excretion between N, P, T1 and T2 are shown in Fig. 1. The urinary protein excretion was significantly less in M2000-treated animals than nontreated control (P < 0.05). Fig. 2 shows the antiproteinuric effect of M2000 therapy compared with patient (P) and piroxicam (T2 ) groups at the end of the experiment (day 84). Here, reduction of proteinuria in T1 versus P and T2 groups were significant (P < 0.05). BUN, serum cholesterol and triglyceride levels were elevated in P and T2 groups when compared with M2000-treated rats (Table 1), but these differences were no significant. Whereas difference between T1 versus P and T2 in reduction of proteinuria was significant (P < 0.05), Table 2. In addition, increase of urea in H group versus P and N groups was significant (P < 0.05), Table 2. Here, decrease in serum determinants concentration and increase in urine determinants (urea and creatinine) levels following M2000 therapy paralleled with the amelioration process of disease.

3.3. Histopathological findings Light microscopic examination of renal tissue revealed the severity of glomerular infiltration of PMN in nontreated (P) and treated group with piroxicam (T2 ) compared with M2000-treated (T1 ) rats (data not shown). Immunofluorescent microscopic investigation of glomeruli revealed that glomerular immune complex deposition was less intense in treated animals with M2000 (T1 ) than controls (P) and treated with piroxicam (data not shown). 3.4. Toxicological findings Data analysis showed no significant differences in the levels of serum determinants (BUN, creatinine, cholesterol, and triglyceride) and urine determinants (protein excretion and creatinine) between the normal group (N) and healthy experimental group (H) challenged with M2000 (Tables 1 and 2). Whereas, significant increase of urine urea in group H compared with normal group (N) revealed the advantage of the use of M2000 in healthy controls (Table 2). Histologic study of kidney specimens obtained from normal group and healthy controls receiving M2000 showed no glomerular changes in group (H) in comparison with normal group (data not shown).

Fig. 1. Time course of the mean values of proteinuria in different groups N, P, T1 and T2 in immune complex glomerulonephritis. N: normal rats (n = 8); P: patient rats (n = 9); T1 and T2 : treated groups with intraperitoneal injections of M2000 (30 mg/kg) and piroxicam (0.3 mg/kg), respectively (n = 2 × 8). Onset of i.p. administration of M2000 and piroxicam to T1 and T2 groups was day 56. Number of i.p. injections (15), injection interval (48 h), and the end of therapeutic protocol was day 84. There was significant difference between M2000-treated group (T1 ) and patient group. P < 0.05 was considered significant.

3.5. Biocompatibility of M2000

Fig. 2. Comparison of antiproteinuric effect of M2000 between groups P, T1 and T2 in the end of experiment (week 12). N: normal (8); P: patient (9); T1 and T2 : treated rats with M2000 (30 mg/kg) and piroxicam (0.3 mg/kg), respectively (2 × 8 = 16). Each bar represents the mean ± S.D. T1 vs. P was significant, “P < 0.05 vs. nontreated.”.

Fig. 3. Anti-BSA antibody titers in groups P, T1 and T2 during the course of acute BSA-nephritis, as determined by an ELISA method. The highest titer occur in P, the intermediate in T2 and the lowest in T1 . Values are means ± S.D. The difference between M2000-treated group (T1 ) and patient group (P) at 12th week was significant. P < 0.05 was considered significant.

Fig. 4 shows the proliferative response of a fibrosarcoma (WEHI-164) cell line to M2000 at different doses (10–200 ␮g/ml) compared with diclofenac, piroxicam, and dexamethasone. The tolerability and biocompatibility of WEHI-

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Table 1 Effect of M2000 on serum determinants Groups

No. of rats

BUN (mg/dl)

N P T1 T2 H

8 9 8 8 8

16 19 18 21 17

± ± ± ± ±

Creatinine (mg/dl)

2 2 2 2 4

0.8 0.9 0.9 0.9 0.8

± ± ± ± ±

0.1 0.1 0.1 0.1 0.1

Triglyceride (mg/dl) 79 76 68 105 89

± ± ± ± ±

9 7 12 30 23

Cholesterol (mg/dl) 59 60 53 64 56

± ± ± ± ±

3 7 7 8 6

Values are expressed as mean ± S.D. N: normal; P: patient; T1 : treated with M2000 (30 mg/kg); T2 : treated with piroxicam (0.3 mg/kg); H: healthy controls receiving M2000 (30 mg/kg). Decrease in serum cholesterol, triglyceride and BUN levels in group T1 compared with patient group was not significant (P < 0.05). Table 2 Effect of M2000 on urine determinants Groups

No. of rats

Protein (mg/24 h)

N P T1 T2 H

8 9 8 8 8

8 44 22 122 6

± ± ± ± ±

Urea (mg/dl)

3 18 10 111 2

939 876 963 895 1284

± ± ± ± ±

300 277 334 252 226

Creatinine (mg/dl) 24 18 25 23 22

± ± ± ± ±

5 5 10 7 4

Values are expressed as mean ± S.D. N: normal; P: patient; T1 : treated with M2000 (30 mg/kg); T2 : treated with piroxicam (0.3 mg/kg); H: healthy controls receiving M2000 (30 mg/kg). Reduction of proteinuria in T1 vs. P and T2 was significant (P < 0.05). Moreover, increase of urea in H group vs. P and N groups was significant (P < 0.05).

164, as a sensitive cell line against increasing amounts of M2000 was very high, whereas 50% of cells died when diclofenac, dexamethasone and piroxicam were added to tissue culture at doses of 25, 80 and 80 ␮g/ml, respectively. M2000 showed no cytotoxic effect compared with steroidal and non-steroidal tested drugs. 3.6. Effect of M2000 on MMP-2 activity Dose response analysis of M2000 on MMP-2 activity is presented in Fig. 5. The inhibitory effect of M2000 at concentrations of 20, 40, 80 and 200 ␮g/ml was significantly more than dexamethasone (P < 0.05), as well as this difference was significant between M2000 and piroxicam at concentration of 200 ␮g/ml Fig. 5. The inhibitory effect of M2000 on MMP-2 activity. Fibrosarcoma cell lines (2 × 104 cells/well) were incubated for overnight with increasing dose of M2000 as described in Section 2. Diclofenac, piroxicam and dexamethasone treated cells were used as controls. M2000 treated and non-treated cells were investigated in triplicate. The inhibitory activity of M2000 paralleled with diclofenac at doses 10, 20, 40 and 200 ␮g/ml, whereas the inhibitory effect of M2000 at concentrations of 20, 40 and 80 ␮g/ml was significantly more than dexamethasone (P < 0.05). Moreover, the inhibitory activity of this novel agent at dose of 200 ␮g/ml was significantly more than dexamethasone and piroxicam (P < 0.05).

(P < 0.05). Whereas, the inhibitory activity of this novel agent paralleled with diclofenac at doses 10, 20, 40 and 200 ␮g/ml. It should be noted that, regarding the similarity of inhibitory effects of M2000 on MMP-9 and MMP-2, thus the results of MMP-2 has been proposed in this study. Fig. 4. Cytotoxic effect of M2000. Proliferative response of fibrosarcoma (WEHI-164) cell line to M2000 at different doses (10–200 ␮g/ml) compared to diclofenac, piroxicam and dexamethasone. LD50 for diclofenac, piroxicam and dexamethasone was 25, 80 and 80 ␮g/ml, respectively. In contrast, WEHI164 as a sensitive cell line showed a high tolerability against increasing amounts of M2000.

4. Discussion In order to produce M2000 (␤-d-mannuronic acid), as a novel designed nonsteroidal anti-inflammatory drug and explore the

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potency of this agent in treatment of experimental glomerulonephritis, this investigation was designed. Glomerulonephritis remains the second or third most common primary renal disease type to progress to end-stage renal failure. This disease type is particularly important because its focus is limited to the kidney and its reversal or stabilization ensures a return to a normal quality of life for the individual. Also, because its highest incidence rate is in childhood and early adulthood, the implications of effective therapy in terms of preventing endstage renal failure costs, benefits not only the individual but also society (Cattran, 2003). Typically, aggressive form of disease is managed by the administration of steroids and a cytotoxic agent, usually cyclophosphamide initially and then azathioprine (O’Callaghan, 2004). Whereas, although nonsteroidal anti-inflammatory drugs (NSAIDS) are used extensively in clinical medicine. In spite of their therapeutic utility, however, they are known to cause significant gastrointestinal and renal toxicities, circumstances that limit their use (Fogo, 2003; Reinhold et al., 2003; Basivireddy et al., 2004). The side effects produced in these organs have been attributed mainly to the inhibitory effect of these drugs on the activity of cyclooxygenase, a key enzyme in prostaglandin synthesis. In addition to this, one of the mechanisms by which NSAIDS induce renal damage is through reactive oxygen species (oxygen free radicals), possibly generated by activated neutrophils and mitochondrials dysfunction (Basivireddy et al., 2004; Stollbergen and Finsterer, 2004). Thus, hemodynamic renal failure may result from drugs that reduce renal prostaglandins and hence renal blood flow and glomerular filtration rate (Perazella, 2003). In the present study, the result of treatment with piroxicam paralleled with the results of administration of other NSAIDS (Basivireddy et al., 2004; Perazella, 2003; Kaiser, 2003), so that, the use of piroxicam could reinforce renal insufficiency in our experimental model. In contrast, M2000 not only did not exacerbate renal damage, but interestingly, this novel designed NSAIDS could significantly diminish renal lesions in experimental model of glomerulonephritis. On the other hand, regarding mesangial cells play a prominent role in renal inflammatory disorder, as well as the role of matrix metalloproteinases (MMPs) in the activation of mesangial cells and with respect to the fact that, increased cell proliferation rates and extracellular matrix accumulation are crucial targets in the therapy of glomerulonephritis (Lenz et al., 2000; Chadban, 2001; Marti, 2002). Therefore, MMP inhibitors provide a new approach to the therapy of inflammation probably even beyond the field of renal disorders (Marti, 2002; Lovett et al., 1992; Harendza et al., 1999). The present research using zymoanalysis method showed that M2000 is a MMP inhibitor. This property as one of characteristics of M2000 can also assist to interpret the mechanism of action of this novel designed NSAID. 5. Conclusion In this study, for the first time we introduced the method of production of M2000 (␤-d-mannuronic acid) and our findings showed that treatment with this drug caused a significant reduction in proteinuria, anti-BSA antibody production as well as suppression of glomerular lesions. M2000 therapy showed

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its potency in the treatment of experiment model of glomerulonephritis. It is the first novel designed NSAID with lowest molecular weight and therapeutic effects on glomerulonephritis which it could be strongly recommended in an extensive scale as a safest drug for decreasing the anti-inflammatory reactions. Acknowledgments M2000 has been awarded its international patent cooperation treaty (WO 2004033472) by funding the University of Munster in Germany. We wish to thank the Minister of science and research of North-Rhine Westphalia of Germany, Dr. Hannelore Kraf for her award to M2000. References Basivireddy, J., Jacob, M., Pulimood, A.B., Balasubramanian, K.A., 2004. Indomethacin-induced renal damage: role of oxygen free radicals. Biochem. Pharmacol. 67, 587–599. Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. Bousnes, R.W., Taussky, A.A., 1945. The calorimetric determination of creatinine by the Jaffe reaction. J. Biol. Chem. 158, 581–591. Bucke, C., 1987. Cell immobilization in calcium alginate. Meth. Enzymol. 135, 175–188. Camp, T.M., Smiley, L.M., Hayden, M.R., Tyagi, S.C., 2003. Mechanism of matrix accumulation and glomerulosclerosis in spontaneously hypertensive rats. J. Hypertens. 21, 1719–1727. Cattran, D.C., 2003. Outcomes research in glomerulonephritis. Semin. Nephrol. 23, 340–354. Chadban, S., 2001. Glomerulonephritis recurrence in the renal graft. J. Am. Soc. Nephrol. 12, 394–402. Evans, R.T., 1968. Manual and automated methods for measuring urea based on a modification of its reaction with diacetyl monoxime and thiosemicarbazide. J. Clin. Pathol. 2, 527–532. Filisetti-Cozzi, T.M., Carpita, N.C., 1991. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197, 157–162. Fogo, A.B., 2003. Quiz page. Acute interstitial nephritis and minimal change disease lesion, caused by NSAIDS injury. Am. J. Kidney Dis. 42, A41–A51. Galambos, J.T., 1967. The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole color of sugars. Anal. Biochem. 19, 119–132. Harendza, S., Schneider, A., Helmchen, U., Stahl, R.A., 1999. Extracellular matrix deposition and cell proliferation in a model of chronic glomerulonephritis in the rat. Nephrol. Dial. Transplant. 12, 2873–2879. Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen activator in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196–202. Hogendoorn, P.C.W., Bruijn, J.A., Gelok, E.W.A., Van Den Broek, L.J.C.M., Fleuren, G.J., 1990. Development of progressive glomerulosclerosis in experimental chronic serum sickness. Nephrol. Dial. Transplant. 5, 100–109. Hricik, D.E., Chung-park, M., Sedor, J.R., 1998. Glomerulonephritis. N. Engl. J. Med. 339, 888–899. Kaiser, A., 2003. Diclofenac caused renal insufficiency. A case illustrating the necessity of pharmaceutical intervention and care. Med. Monatsschr Pharm. 26, 384–388. Lenz, O., Elliot, S.J., Stetler-Stevenson, W.G., 2000. Matrix metalloproteinases in renal development and disease. J. Am. Soc. Nephrol. 11, 574–581. Lovett, D.H., Johnson, R.J., Marti, H.P., Martin, J., Davies, M., Couser, W.G., 1992. Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis. Am. J. Pathol. 141, 85–98. Marti, H.P., 2002. The role of matrix metalloproteinases in the activation of mesangial cells. Transpl. Immunol. 9, 97–100.

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Mirshafiey, A., Rehm, B.H., Sahmani, A.A., Naji, A., Razavi, A., 2004. M-2000, as a new anti-inflammatory molecule in treatment of experimental nephrosis. Immunopharmacol. Immunotoxicol. 26, 611–619. Mirshafiey, A., Matsuo, H., Nakane, S., Rehm, B.H., Koh, C.S., Miyoshi, S., 2005a. Novel immunosuppressive therapy by M2000 in experimental multiple sclerosis. Immunopharmacol. Immunotoxicol. 27, 255–265. Mirshafiey, A., Cuzzocrea, S., Rehm, B., Mazzon, E., Saadat, F., Sotoude, M., 2005b. Treatment of experimental arthritis with M2000, a novel designed non-steroidal anti-inflammatory drug. Scand. J. Immunol. 61, 435–441. O’Callaghan, C.A., 2004. Renal manifestations of systemic autoimmune disease: diagnosis and therapy. Best Pract. Res. Clin. Rheumatol. 18, 411–427. Perazella, M.A., 2003. Drug- induced renal failure: update on new medications and unique mechanisms of nephrotoxicity. Am. J. Med. Sci. 325, 349–362. Quigg, R.J., 2004. Complement and autoimmune glomerular diseases. Curr. Dir Autoimmun 7, 165–180. Rehm, B.H.A., 1998. Alginate lyase from pseudomonas aeruginosa CF1/M1 prefers the hexameric oligomannuronate as substrate. FEMS Microbiol. Lett. 165, 175–180. Rehm, B.H., Valla, S., 1997. Bacterial alginates: biosynthesis and applications. Appl. Microbiol. Biotechnol. 48, 281–288. Reinhold, S.W., Fischereder, M., Riegger, G.A., Kramer, B.K., 2003. Acute renal failure after administration of a single dose of a highly selective COX-2 inhibitor. Clin. Nephrol. 60, 295–296.

Rougier, J.P., Ronco, P., 2003. Pathogenesis of immune glomerulonephritis. Rev. Prat. 53, 1998–2004. Sanders, J.S., van Goor, H., Hanemaaijer, R., Kallenberg, C.G., Stegeman, C.A., 2004. Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis. Nephrol. Dial. Transplant. 19, 1412– 1419. Skjak-Braek, G., Grasdalen, H., Larsen, B., 1986. Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydrate Res. 15, 239–250. Stollbergen, C., Finsterer, J., 2004. Side effects of conventional nonsteroidal anti-inflammatory drugs and celecoxib: more similarities than differences. South Med. J. 97, 209–219. Tarzi, R.M., Cook, H.T., 2003. Role of Fcgamma receptors in glomerulonephritis. Nephron Exp. Nephrol. 95, e7–e12. Vilim, V., 1985. Colorimetric estimation of uronic acids using 2hydroxydiphenyl as a reagent. Biomed. Biochim. Acta 44, 1717–1720. Vinen, C.S., Oliveira, D.B.G., 2003. Acute glomerulonephritis. Postgrad. Med. J. 79, 206–213. Yamamoto, T., Kihara, I., Morita, T., Oite, T., 1978. Bovine serum albumin (BSA) nephritis in rats. I. Experimental model. Acta Pathol. Jpn. 28, 859–860. Zhang, Z.G., Liu, X.G., Chen, G.P., Zhang, X.R., Guo, M.Y., 2004. Significance of MMP-2 and TIMP-2 mRNA expressions on glomerular cells in the development of glomerulosclerosis. Med. Sci. J. 19, 84–88.