Effects of low molecular weight chitosans on aristolochic acid-induced renal lesions in mice

Food Chemistry 129 (2011) 1751–1758

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Effects of low molecular weight chitosans on aristolochic acid-induced renal lesions in mice Ya-Min Chang a, Chen-Tien Chang a, Tzu-Chuan Huang b, Shih-Ming Chen c, Jen-Ai Lee b,⇑⇑, Yun-Chin Chung a,⇑ a b c

Department of Food and Nutrition, Providence University, Taichung, Taiwan Department of Pharmaceutical Analysis, School of Pharmacy, Taipei Medical University, Taipei, Taiwan Department of Clinical Pharmacy, School of Pharmacy, Taipei Medical University, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 24 January 2011 Received in revised form 17 June 2011 Accepted 23 June 2011 Available online 30 June 2011 Keywords: Bamboo chitosanase Aristolochic acid Low molecular weight chitosans Renal histopathology Renal lesions

a b s t r a c t This study investigated the effects of low molecular weight chitosan (LMWC) on the renal functions for aristolochic acid (AA)-induced renal lesions in C3H/He mice. Commercial crab shell chitosan (molecular mass of 1100 kDa) was hydrolysed using bamboo chitosanase at pH 3.5 and 50 °C for 18 h. LMWC (with molecular mass of 29 kDa) was isolated from the hydrolysate through 50% methanol fractionation. The levels of urinary microalbumin, urinary-N-acetyl-beta-D-glucosaminidase, serum creatinine, and blood urea nitrogen were higher in the AA induced mice than those in the control group, while the rate for creatinine clearance was lower in the AA induced mice. Oral administration of LMWC could suppress the elevation of urinary and blood nitrogen concentrations induced by AA treatment. Renal histopathological examination revealed that AA-induced renal alterations were reverted effectively by the treatment of LMWC at three tested doses, especially at the moderate dose of 500 mg/kg BW. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Rouget (1859) first prepared chitosan from heat-alkaline deacetylation of chitin. With a unique structure, (1,4)-2-amino-2deoxy-b-D-glucan, chitosan proved to have many special properties such as biocompatibility, biodegradability, non-toxicity, stimulation of plant growth and maturation (Kumar, 2000). However, its high molecular weight (HMWC), between 500 kDa and 1000 kDa, and its high viscosity, has limited the commercial application of chitosan. Depolymerized chitosan products (i.e. low molecular weight chitosan (LMWC) and chitooligosaccharides (COS)) have overcame these limitations. LMWC and COS have shown improved antitumor and antimicrobial characteristics, superior antioxidation, and a greater capacity to stimulate the immune system and reduce cholesterol, when compared with HMWC (Kendra & Hadwiger, 1984; Tsai, Wu, & Su, 2000; Xia, Liu, Zhang, & Chen, 2011). Low molecular weight chitosan (LMWC) and chitooligosaccharides (COS) are produced through chemical or enzymatic hydroly-

⇑ Corresponding author. Address: Department of Food and Nutrition, Providence University, 200 Chungchi Rd., Taichung 433, Taiwan. Tel.: +886 4 26328001x15345; fax: +886 4 26530027. ⇑⇑ Co-corresponding author. Address: Department of Pharmaceutical Analysis, School of Pharmacy, Taipei Medical University, No. 250, Wu-Hsing St., Taipei 110, Taiwan. Tel.: +886 2 2736 1661x6125. E-mail addresses: [email protected] (J.-A. Lee), [email protected] (Y.-C. Chung). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.044

sis of chitosan chains. Compared to the chemical hydrolysis of chitosan chains, enzymatic hydrolysis is less susceptible to pH and heat. Enzymatic hydrolysis also dramatically reduces reaction time from 20 days to 48 h when papain is used to replace 0.6 M HCl (Terbojevich, Cosani, & Muzzarelli, 1996). Researchers have used commercial enzymes, such as cellulase, papain, pepsin, ficin, lipase, stem bromelain, to degrade chitosan in the production of LMWC or COS (Hung, Chang, Sung, & Chang, 2002; Lee, Xia, & Zhang, 2008; Muzzarelli, Terbojevich, Muzzarelli, & Francescangeli, 2002; Roncal, Oviedo, López de Armentia, Fernández, & Villarán, 2007; Tsai et al., 2000). Terbojevich et al. (1996) compared the effects of papain, lipase, and lysozyme on chitosan and found that papain was the most capable for the production of LMWC. Approximately one quarter of all dialysis patients suffer from renal disease. Regular drug therapy for renal disease often induces complicated systemic side effects. Researchers have reported renal injury associated with the use of several herbs. The best known herb-induced chronic kidney disease (CKD) is aristolochic acid nephropathy (Shaohua et al., 2010; Zhou et al., 2010). Aristolochic acid (AA) is the main active component in plants of the Aristolochia species (Zhang et al., 2006). AA has been a traditional Chinese herbal remedy with potent diuretic activity. Toxicological studies have revealed that AA is associated with renal tubular damage, tubulointerstitial fibrosis, and even DNA adducts of renal tissue (Attaluri et al., 2010; Zhou et al., 2010). Research has suggested that chitosan supplements might be an effective treatment for renal failure (Jing, Li, Ji, Takiguchi, &

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Yamaguchi, 1997). A calcium carbonate and chitosan supplement decreased plasma and inorganic urea phosphate in aged cats with moderate chronic renal failure (Wagner, Schwendenwein, & Zentek, 2004), and chitooligosaccharides aided in recovery from glycerol-induced acute renal failure (Yoon et al., 2008). In the treatment of renal failure, LMWC served as a phosphate-binding agent or renal targeting carrier (Yuan et al., 2007; Savica, Santoro, Monardo, Mallamace, & Bellinghieri, 2009). This study prepared LMWC using bamboo chitosanase hydrolysis of chitosan and investigated the effects of LMWC on the factors influencing renal function and renal histopathology for aristolochic acid-induced renal lesions in C3H/He mice. 2. Materials and methods 2.1. Chemicals Dextran molecular weight standards (MW = 80,900; MW = 48,600; MW = 23,800; MW = 11,600; MW = 3260; MW = 1270) were obtained from Fluka Company (Buchs, Switzerland). Superose 12 HR (10/30) columns were purchased from GE Healthcare BioSciences AB (Uppsala, Sweden). Crab shell chitosans (84% deacetylation; approximately 1100 kDa) was provided by Ohka Enterprises C. Ltd. (Kaohsiung, Taiwan). Aristolochic acid sodium salt (AANa), 3-methyl-2-benzothiazolone hydrochloride (MBTH), bovine serum album (BSA), buffered neutral formalin solution, cimetidine, creatinine, 4-methylumbelliferyl N-acetyl-b-D-glucosaminide (4-MUNAG), N-acetyl-b-D-glucosaminide (NAG), paraformaldehyde, periodic acid, sodium bisulphate, sodium heparin, and xylene were purchased from Sigma–Aldrich Fine Chemicals Inc. (St. Louis, MO, USA). Entellan and a urease assay kit were obtained from MERCK (Darmstadt, Germany). The BioRad protein assay kit was purchased from Bio-Rad (Richmond, CA, USA). All other chemicals were reagent grade or purer. 2.2. Preparation of chitosanase from bamboo shoots To obtain chitosanase, the bamboo (Bambusa oldhamii) shoots were coated with chitosan and stored at room temperature (25 ± 2 °C) for 42 h. The edible part of the bamboo shoots was lyophilised and ground into a powder. Crude chitosanase was extracted by stirring the bamboo shoot powder (10 g) into 200 ml of sodium acetate buffer (0.05 mM, pH 4.5) at 4 °C for 45 min. Crude chitosanase was fractionated through the addition of (NH4)2SO4. A precipitate formed at 80% saturation of (NH4)2SO4 was collected by centrifugation (17,540g, 10 min) and dissolved in 10 ml of 0.05 M sodium acetate buffer (pH 4.5). After centrifugation (17,540g, 20 min), a supernatant was used to digest the chitosan. 2.3. Determination of chitosanase activity A mixture of 0.1 ml of 0.5% chitosan in 0.1 M of sodium acetate buffer (pH 4.0), 0.15 ml of 0.1 M sodium acetate buffer (pH 4.0), 0.1 ml of H2O and 0.05 ml of chitosanase solution, for a total volume of 0.4 ml, was incubated at 37 °C for 30 min. The reaction was stopped by adding 0.8 ml of Reagent A and 0.8 ml of Reagent B (Reagent A: 40 g of Na2CO3, 16 g of glycine and 450 mg of CuSO45H2O in 1 l of H2O; Reagent B: 1.5 g of neocuproine hydrochloride in 1 l of H2O) and incubating the mixture at 100 °C for 14 min (Dygert, Li, Florida, & Thoma, 1965). The amount of neocuproine complex formed was measured by determining the absorbance at 450 nm using a spectrophotometer (Hitachi U2001, Tokyo, Japan). One unit of enzyme was defined as the amount of enzyme required to liberate 1 lmol of D-glucosamine per minute.

2.4. Preparation of LMWC Chitosan (9 g) in 200 ml of 4.5% acetic acid (final pH 3.8, adjusted by 0.425 M NaHCO3) was digested with 200 ml (180 units) of partially purified bamboo chitosanase in a shaking incubator (125 rpm) at 50 °C for 18 h. Following hydrolysis, the hydrolysate was boiled for 15 min to denature the enzyme, which was then removed by filtration. The filtrate was adjusted to pH 7.0 with 0.425 M NaHCO3, and then centrifuged (5000g, 15 min). The supernatant was mixed with an equal volume of methanol. After removing a small amount of precipitate by centrifugation (5000g, 15 min), the supernatant was concentrated by evaporation under reduced pressure until the final volume was 10% of the initial volume. The supernatant was then lyophilised. The lyophilised LMWC product was thoroughly washed twice with 1000 ml of 95% ethanol using centrifugation (5000g, 15 min). Finally, the washed LMWC was suspended in a small volume of water and lyophilised. 2.5. Determination of molecular mass The molecular mass of LMWC was determined by gel filtration on an FPLC System (Amersham Pharmacia Biotech AB, Uppsala, Sweden) with a Superose 12 HR (10/30) column. The LMWC powder (5 mg) was resolved with 2 ml of 0.05 M sodium acetate buffer (pH 4.5), and the supernatant was collected by centrifugation at 6000g for 15 min. The Superose 12 HR (10/30) column was equilibrated with 0.1 M sodium acetate buffer (pH 4.5) at a flow rate of 30 ml/h. Next, 0.1 ml of LMWC solution was added to the column and eluted with the equilibrium buffer. Each of the collected fractions (0.5 ml) was analysed for D-glucosamine concentration using the MBTH colorimetric method (Tsuji, Kinoshita, & Hoshino, 1972). Calibration of the column was performed according to dextran standards (molecular masses: 80.9, 48.6, 23.8, 11.6, 3.26, and 1.27 kDa). A calibration curve was obtained by plotting the elution volume of dextran standards versus the log of the molecular mass. The molecular mass of LMWC was calculated from the calibration curve. 2.6. Animal treatment Sixty-four 6-week-old female C3H/He mice, were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). Eight mice were housed per cage under controlled environmental conditions (25 ± 2 °C, 65 ± 5% relative humidity, 0700–1900 h lighting period). Standardised food (TMI, USA) and water were freely available. All animals received human care according to the guidelines of the Guidebook for the Care and Use of Laboratory Animals (Yu et al., 2004). The animal research ethics committee at Taipei Medical University, Taipei, Taiwan, approved the study protocol. The mice were divided into 8 groups of 8 animals each after 1 week of acclimation (Table 1). The control group was injected with 0.1 ml normal saline for 5 days (day 1–5) and retained as C group. The AA group was injected with a dosage of 0.1 ml AA (10 mg/kg BW/day) for 5 days (day 1–5). The SH, SM and SL groups were gavaged with 0.2 ml LMWC at doses of 1500, 500, and 250 mg/kg BW/day for 14 days (day 6–19), respectively, following saline treatment for 5-days. The AH, AM, and AL groups were treated with 0.2 ml LMWC at doses of 1500, 500, and 250 mg/kg BW/day for 14 days (day 6–19), respectively, following AA treatment for 5-days. Following the completion of the experiment, the animals were sacrificed under pentobarbital (50 mg/kg BW) anaesthesia and a blood sample was collected by intracardiac puncture with a syringe. A thorough necropsy was carried out on all animals. Prior to sacrifice, mice were placed in rodent metabolic cages for urine collection.

Y.-M. Chang et al. / Food Chemistry 129 (2011) 1751–1758 Table 1 Grouping of animal and treatment. Groupa

Treatments (mg/kg BW/day)

C AA SH SM

Saline (i.v.) for 5 days 10 (i.v.) for 5 days 1500 (P.O) after 5 days saline treatment 500 (P.O) after 5 days saline treatment

SL AH AM AL

Control (saline) Aristolochic acid (AA) Saline + LMWC (high dose) Saline + LMWC (middle dose) Saline + LMWC (low dose) AA + LMWC (high dose) AA + LMWC (middle dose) AA + LMWC (low dose)

250 (P.O) after 5 days saline treatment 1500 (P.O) after 5 days AA treatment 500 (P.O) after 5 days AA treatment 250 (P.O) after 5 days AA treatment

a Animals were divided into 8 groups of 8 animals each. Group C was injected with 0.1 ml isotonic saline for 5 days and retained as the control. Group AA was injected with a dosage of 0.1 ml AA (10 mg/kg BW/day) for 5 days and retained as the AA treated. Groups, SH, SM, and SL were treated with 0.2 ml LMWC at doses of 1500, 500 and 250 mg/kg BW/day, respectively following saline treatment. Groups AH, AM, and AL were treated with 0.2 ml LMWC with doses of 1500, 500, and 250 mg/kg BW/day, respectively, following AA treatment.

2.7. Preparation of urine, blood, and kidney samples Twelve-hour urine samples were collected by a metabolic cage (Tokiwa Co. Ltd., Tokyo) individually and centrifuged (700g, 15 min), and the clear supernatant was stored at 20 °C prior to analysis. Blood samples were centrifuged (700g, 15 min) and the serum was stored at 20 °C until assayed. Blocks of renal tissue were fixed in 10% buffered formaldehyde solution and embedded in paraffin for routine histological examination. The paraffin was cut into 6 lm sections, stained with haematoxylin and eosin (H&E), and examined under light microscopy at a magnification of 200 (Optima G-300, Leica DM IRB, Wetzlar, Germany). 2.8. Serum and urinary creatinine assays Serum and urinary creatinine levels were measured using a high-performance liquid chromatographic method, as previously described (Lee et al., 2005), and expressed in mmol/l. A 0.1 mM cimetidine in a 0.01 M HCl aqueous solution served as the internal standard. Samples were vortex-mixed and centrifuged at 700g for 10 min. The supernatants were analysed by HPLC using a Biosil ODS (C18 column) (4.6  250 mm I.D., particle size 5 lm) (Biosil Chemical Co., Ltd., Taipei, Taiwan) with an L-4200 UV–Vis detector (Hitachi, Tokyo, Japan) monitored at 234 nm. The mobile phase consisted of acetonitrile and 100 mM of a potassium phosphate buffer solution containing 30 mM sodium lauryl sulphate adjusted to pH 3.0 with o-phosphoric acid (7/12, v/v). The flow rate was 0.7 ml/min. The creatinine clearance (Ccr), an effective index for expressing the glomerular filtration rate (GFR), was calculated using the following equation (Hsu, Wana, Hsu, Lin, & Liu, 2008):

Ccr ¼

Ucr  UV 1000 1   SCr BW 720

Ccr is creatinine clearance (ml/min/kg BW), Ucr is urinary creatinine (mg/dl), UV is urine volume (ml), Scr is serum creatinine (mg/dl), BW is body weight (g), 720 is time (min). 2.9. Determination of urinary protein and N-acetyl-b-Dglucosaminidase (NAG, EC 3.2.1.30) activity Urinary excretion of protein was quantified using the BioRad protein assay kit with bovine serum albumin as the standard. Bradford reagent was added to the standard or urine samples. Optical density was measured at 595 nm and the concentration of protein

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was calculated from a bovine serum albumin standard curve (Bradford, 1976). NAG activity was determined according to Leaback and Walker (1961). In this assay, NAG reacted with the 4-methylumbelliferylN-acetyl-b-glucosaminide (4-MU-NAG) and the release of fluorescence was quantified as a concentration of NAG. 2.10. Determination of blood urea nitrogen (BUN) concentration The concentration of BUN was assessed with a urease assay kit, in which urea had been hydrolysed into ammonia and carbon dioxide using urease. The decrease of NADH was monitored by reading A340 under the glutamate dehydrogenase catalysis of ammonia and a-ketoglutaric acid to L-glutamic acid (Kaltwasser & Schlegel, 1966). 2.11. Histopathological examination The required animal tissue was embedded in paraffin, sectioned at 3–5 lm in thickness, and stained with haematoxylin and eosin (H&E) and Masson’s trichrome (TRI). A histopathological evaluation was performed on the kidneys of each of the test animals in the control and treatment groups. The severity of lesions was graded according to the methods described by Shackelford, Long, Wolf, Okerberg, and Herbert (2002). The degrees of lesions were graded histopathologically from one to five depending on severity (1 = minimal; 2 = slight; 3 = moderate; 4 = moderately severe; 5 = severe/high). The sum of scores for each sample, including acute tubular necrosis, hyaline casts, tubular dilatation, and infiltration, was defined as the tubulointerstitial histological score (TIHS) according to Sato et al. (2004). 2.12. Statistical analysis All data were expressed as mean ± S.E.M. for the eight mice in each group. The significance of differences among groups was analysed by one-way analysis of variance (ANOVA) and with Duncans’ multiple range test using the one way program in the SPSS 10.0 package. Differences were considered significant at p < 0.05. 3. Results 3.1. Chitosanase from bamboo shoots Crude chitosanase was extracted from the bamboo shoot powder, and the partially purified chitosanase with specific activity of 78.2 mU/mg was obtained by sequential step of ammonium sulphate fractionation. To produce LMWC, commercial crab shell chitosan (molecular mass of 1100 kDa) was hydrolysed using bamboo chitosanase. 3.2. Molecular mass of LMWC obtained by gel filtration This study determined the molecular mass distribution of LMWC by gel filtration on a Superose 12 HR (10/30) column using dextran with various molecular masses as a calibration marker. As shown in Fig. 1, this experiment eluted unhydrolysed high molecular weight chitosan (1100 kDa), from the column with a major peak at fraction 18 (9 ml, elution volume) (Fig. 1A). The calibration curve obtained from the elution volumes of dextran standards was shown in Fig. 1B. The LMWC obtained after 18 h of hydrolysis, catalysed by bamboo chitosanase was fractionated into a major peak between fractions 32 and 38 (16–19 ml) and several minor peaks

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Y.-M. Chang et al. / Food Chemistry 129 (2011) 1751–1758 Table 2 Change of body weight during experimental period.

0.5

Absorbance at 650 nm

A

Group*

(0 hr)

0.4

Day 7

C AA SH SM SL AH AM AL

0.3

0.2

Day 5

Day 19 ab

20.46 ± 1.0 20.16 ± 0.73 20.24 ± 0.98 20.06 ± 0.87 20.26 ± 0.87 20.28 ± 1.13 20.57 ± 0.99 20.93 ± 0.44

21.56 ± 0.80 19.98 ± 0.83 c 21.92 ± 0.84 a 21.77 ± 0.9 a 21.62 ± 0.99 ab 19.76 ± 1.35 c 20.59 ± 1.58 bc 20.85 ± 0.66 abc

21.90 ± 2.35 17.60 ± 0.88 21.98 ± 0.91 22.17 ± 1.11 22.06 ± 0.80 19.16 ± 1.68 19.81 ± 2.38 18.46 ± 1.25

a c a a a cb b cb

*

Animals were divided into 8 groups of 8 animals each. Group C was injected with 0.1 ml isotonic saline for 5 days and retained as the control. Group AA was injected with a dosage of 0.1 ml AA (10 mg/kg BW/day) for 5 days and retained as the AA treated. Groups, SH, SM, and SL were treated with 0.2 ml LMWC at doses of 1500, 500, and 250 mg/kg BW/day, respectively following saline treatment. Groups AH, AM, and AL were treated with 0.2 ml LMWC with doses of 1500, 500, and 250 mg/kg BW/day, respectively, following AA treatment.

0.1

0.0 0

10

20

30

40

50

60

70

3.26

B

Fraction number 0.7

Absorbance at 490 nm

0.6

experiment (data not shown). The AH, AM, and AL groups lost an average of 5.5%, 3.7%, and 11.8% body weight, respectively, by the end of the experiment (Table 2).

80.9 0.5

1.27

11.6 23.8

0.4

3.4. Effect of LMWC on the clinical symptom of mice

48.6

0.3

All of the treated mice survived the experimental period. Before and after the test period, no significant changes were observed in body weight among the C, SH, SM, or SL groups (p > 0.05, data not shown). No significant clinical signs indicated treatment with LMWC. However, oral administration of LMWC for 14 days did not significantly change the test parameters (p > 0.05), including urinary microalbumin, urinary NAG level, serum creatinine, serum creatinine clearance level, and blood urea nitrogen (Figs. 2–5). These observations indicate that the LMWC prepared in this study is generally safe in mice at limited dosages (250–1500 mg/kg BW/day), and that LMWC has no significant adverse effects on C3H/He mice.

0.2 0.1 0.0 0

10

20

30

40

50

60

70

Fraction number 0.24

C

Absorbance at 650 nm

0.22

(18hr)

0.20 0.18 0.16

3.5. Effect of the LMWC on urinary microalbumin levels in C3H/He mice Urinary microalbumin levels in the mice increased significantly with an intravenous injections of AA at day 19 (p < 0.05; Fig. 2), compared to the control. However, urinary microalbumin levels also increased significantly with an intravenous injection of

0.14 0.12 0.10 0.08

50

0.04 0

10

20

30

40

50

60

70

Fraction number Fig. 1. Gel filtration of LMWC on a Superose 12 HR column. (A) Unhydrolysed native chitosans (1100 kDa), (B) standard molecular weight dextrans (numbers on the peak indicate dextran size markers in kDa). (C) LMWC obtained from chitosans hydrolysis catalysed by bamboo chitosanase for 18 h.

(Fig. 1C). The molecular mass of LMWC was estimated to be approximately 29 kDa. 3.3. Body weight In this 19 day experiment, the control, SH, SM, and SL mice gained 10%, 9%, 10%, and 9% body weight, respectively. The body weight of the AA group decreased continually throughout the test period, with an average loss of 14.2% body weight by the end of

Urinary microalbumin (mg/dl)/Ucr (mg/dl)

0.06

a

a

a

40

b

30

c

c

c

c

20

10

0 C

AA SH SM SL AH AM AL

Mice group Fig. 2. Effect of oral administration of LMWC on urinary microalbumin in normal, LMWC and AA-treated mice. The data are means ± SD for each group (n = 8). Bars with different letters are significantly different (p < 0.05). (j) C group, (h) AA group, ( ) SH group, ( ) SM group, ( ) SL group, ( ) AH group, ( ) AM group and ( ) AL group.

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Y.-M. Chang et al. / Food Chemistry 129 (2011) 1751–1758

140 a b c

400 d

d

a

a b

120

d

d

b c

c

c

BUN (mg/dl)

Urinary NAG (U)/Ucr (mg/dl)

500

300

200

c

b

b

c

100 80 60 40

100 20

0

C

AA SH SM SL

AH AM

0

AL

C

AA SH

Mice group

SM SL AH AM

AL

Mice group

Fig. 3. Effect of oral administration of LMWC on urinary NAG activity in normal and AA-treated mice. The data are means ± SD for each group (n = 8). Bars with different letters are significantly different (p < 0.05). (j) C group, (h) AA group, ( ) SH group, ( ) SM group, ( ) SL group, ( ) AH group, ( ) AM group and ( ) AL group.

Fig. 5. Effect of oral administration of LMWC on blood urea nitrogen in normal and AA-treated mice. The data are means ± SD for each group (n = 8). Bars with different letters are significantly different (p < 0.05). (j) C group, (h) AA group, ( ) SH group, ( ) SM group, ( ) SL group, ( ) AH group, ( ) AM group and (Q) AL group.

3.6. Effect of the LMWC ingestion on urinary NAG levels

Plasma creatinine (mg/dl)

0.35 a 0.30

b c

c

c

b

b

A

c

0.25 0.20 0.15 0.10 0.05 0.00

C

AA SH SM SL AH

Fig. 3 illustrates the effect of LMWC ingestion on the urinary NAG level. The AA group had a higher NAG level (388.9 ± 18.8 U/ Urinary creatinine (Ucr, mg/dl)) than the control group had (122.8 ± 7.3 U/Ucr (mg/dl)). Compared to the control, the urinary NAG levels did not change significantly in the SH (156.2 ± 11.9 U/ Ucr (mg/dl)), SM (162.1 ± 23.3 U/Ucr (mg/dl)), or SL groups (113.4 ± 13.0 U/Ucr (mg/dl)). Compared with the AA group, the level of urinary NAG in the AH (297.7 ± 30.2 U/Ucr (mg/dl)) and AM (240 ± 27.7 U/Ucr (mg/dl)) groups significantly decreased at day 19 (p < 0.05). 3.7. Effect of the LMWC ingestion on serum creatinine level and creatinine clearance level

AM AL

Mice group

Ccr (ml/min/kg B.W.)

12

a

a b

10

a b c

a b c d

b c d

e

8

B d e

6 4 2 0

C

AA SH SM SL

AH AM

AL

Mice group Fig. 4. Effect of oral administration of LMWC on serum creatinine (A) and creatinine clearance (B) in normal and AA-treated mice. The data are means ± SD for each group (n = 8). Bars with different letters are significantly different (p < 0.05). (j) C group, (h) AA group, ( ) SH group, ( ) SM group, ( ) SL group, ( ) AH group, ( ) AM group and ( ) AL group.

1500 mg LMWC/kg BW/day or 250 mg LMWC/kg BW/day after AA treatment (AH or AL group in Fig. 2). The increase in urinary microalbumin levels, induced by an injection of AA, diminished with the oral administration of 500 mg LMWC/kg BW/day after 14 days (AM group in Fig. 2).

The AA group indicated a higher serum creatinine level (0.28 ± 0.02 mg/dl) than the control group did (0.08 ± 0.01 mg/dl) at day 19 (Fig. 4A). The serum creatinine level of the AH, AM, and AL groups was 0.18 ± 0.006 mg/dl, 0.17 ± 0.02 mg/dl, and 0.20 ± 0.02 mg/dl, respectively. Oral administration of LMWC diminished serum creatinine levels, whether the dose was high, medium, or low. At day 19, the creatinine clearance level (2.2 ± 0.3 ml/min/kg BW) dramatically decreased after the AA group received an intravenous injection, when compared with the control group (10.3 ± 0.8 ml/min/kg BW) (Fig. 4B). The creatinine clearance levels for the AH, AM, and AL groups were 5.1 ± 0.8 ml/min/kg BW, 6.2 ± 0.9 ml/min/kg BW, and 4.2 ± 0.7 ml/min/kg BW, respectively. These levels were significantly higher than those in the AA group (p < 0.05). 3.8. Effect of the LMWC ingestion on blood urea nitrogen concentration Fig. 5 shows the blood urea nitrogen (BUN) concentration at day 19. Blood urea nitrogen was raised significantly in the AA group. No significant difference in the BUN concentration was evident in the C, AH, AM, and AL groups. The BUN levels in each of those groups were all significantly lower than that of the AA group (p < 0.05). 3.9. Morphological findings In the control group, kidney lesions appeared in only one mouse. Infiltration of mononuclear cells in the kidney character-

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ised the lesion and the lesion showed a minimal level of change. This study did not regard the lesion as a treatment-related lesion (Fig. 6A). In the saline control mice, all renal tissue samples were histologically normal (Fig. 6B–D). Obviously, AA exerted a strong nephrotoxic effect on the mice, due to the fact that observations revealed renal lesions and microscopy showed moderately severe levels of change at day 19 (Fig. 6E). Acute tubular necrosis, hyaline casts, tubular dilatation, infiltration of inflammatory cells at the interstitium and perivascular, crystal formation in the lumina of the cortex tubules, and regeneration of the renal tubules characterised these changes. Large aggregates of characteristic brown crystals distended the distal tubules. The formation of brown crystal maybe caused by bleed, blood stasis or blood cell precipitation. Injury to renal tubules due to aristolochic acid caused acute tubular necrosis leading to tubular lesions. In addition, a distinct increase

in the number of leucocytes within interstitial vessels in and around the injured renal areas was observed, and the degenerative cells often remained attached to the basement membrane. The regeneration of renal tubules was initiated to restore the necrotic epithelium; however, this study found no glomerular lesions. Treatment with LMWC partially reversed the tubulointerstitial nephritis (Fig. 6F–H); however, the severity of toxic nephropathy in AL (moderately severe) was higher than AM (moderate) and AH (moderate). The results of the study indicate that the severity of lesions was correlated to dosage. The morphological changes in the lesions consisted of acute tubular necrosis, hyaline casts, tubular dilatation, infiltration of inflammatory cells at the interstitium and perivascular, crystal formation in the lumina of the cortex tubules, and regeneration of renal tubules. The severity of the lesions observed in the AM and AH groups was relatively mild com-

Fig. 6. Staining with haematoxylin and eosin (H&E). Light micrography of renal tissue of normal mice after oral administration of water (A) or LMWC at a high (B), middle (C), and low (D) dose for 14 days (day 19) and AA-treated mice after oral administration of water (E) or LMWC at a high (F), middle (G), and low (H) dose for 14 days (day 19). (#): normal, ( ): regeneration, (&): acute tubular necrosis, (N): hyaline cast, open circle: brown crystals.

Y.-M. Chang et al. / Food Chemistry 129 (2011) 1751–1758

pared to the severity of lesions observed in the AA and AL groups (lesions in AA and AL were graded as moderately severe). At day 19, an increased number of regenerating tubules in AH and AM characterised the progression of lesions. Attenuation of the lining of the epithelium, a wide separation of nuclei, and mitotic indica-

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tive of tubular epithelial necrosis and regeneration were noted. The glomerular of the treatment animals was unaffected. The study detected interstitial fibrosis in the sample (LMWC) treatment group. Interstitial fibrosis was predominant in AH and AM animals. Histological examination revealed a minimal change in interstitial fibrosis (Fig. 7A and B), confirmed by Masson’s trichrome (TRI) staining. Fibrosis was localised in the focal tubular interstitial distribution. Examination of other kidney tissue (C, AA, SH, SM, SL, and AL) samples revealed no significant interstitial fibrosis (data not shown). Tubular regeneration and interstitial fibrosis were significant in AH and AM groups suggesting that LMWC had been effective in repairing AA-induced renal lesions. 3.10. Changes of tubulointerstitial histological scores This study calculated the median scores for the degeneration of tubular atrophy, mononuclear cell infiltration into the interstitium, and interstitial fibrosis using data collected from each group (Fig. 8). The AA group had the highest score. No significant change was observed between the control, SH, SM, and SL groups (p > 0.05). The histological scores indicate that renal damage in the AH, AM, and AL groups was significantly less than that of the AA group. 4. Discussion

Fig. 7. Staining with Masson’s trichrome (TRI). Light micrography of renal tissue of normal mice after oral administration of LMWC at a high (A) and middle (B) dose for 14 days (day 19).

d

C

a

Mice group

AA SH

d

SM

d

SL

d c

AH

c

AM

b

AL

0

1

2

3

4

5

6

Tubulointerstitial histological score Fig. 8. Quantitative analysis of lesions in renal tissue of normal mice after oral administration of water (C) or LMWC at a high (SH), middle (SM), and low (SL) dose for 14 days (day-19) and AA-treated mice after oral administration of water (AA) or LMWC at a high (AH), middle (AM), and low (AL) dose for 14 days (day 19). Bars with different letters are significantly different (p < 0.05). (j) Tubular atrophy, (P) cell infiltration and ( ) interstitial fibrosis.

Researchers have been studying nephropathy due to aristolochic acid (AA) for a considerable length of time. For this reason, this study chose AA for the inducement of renal lesions. As expected, results of histopathological examination indicated that AA causes severe kidney damage and nephrotoxic effects, consisting mainly of tubular atrophy and cell infiltration in C3H/He mice. Oral administration of LMWC for 14 days reduced renal destruction, which may explain why treatment with LMWC decelerated weight loss due to AA. A number of materials (compounds) in the test drug or metabolites of the test drug caused acute tubular necrosis leading to tubular lesions. In this study, AA treatment did not cause remarkable glomerular change; therefore, it is clear that treatment with LMWC partially reversed renal damage. As the animals were injected with AA for 5 consecutive days, it was expected that the most severe renal damage would appear at day 5 or day 6. The self-repairing system might have restored a portion of the damage by day 19, which was 14 days following treatment with AA. The regeneration of renal tubules shown in the AH, AM, and AL groups was initiated to restore necrotic epithelium. The tubular basement membrane was intact, therefore it was determined that regeneration of tubular epithelium was possible. As serum levels of urea and creatinine are important indicators of renal function, a reduction in these levels suggests that renal functionality had improved. The creatinine clearance value also serves as an important index of the excretory function of kidneys (Takashi et al., 2004). Data related to biochemical analysis has revealed that the levels of urinary microalbumin, urinary-NAG, serum creatinine, and BUN were higher in the AA-treated groups than in the control group, but Ccr was lower in the AA-treated groups than in the control group. BUN level is linked to the total body condition of mice and may reflect the severity of tubular necrosis. The BUN decrease suggests the recovery of body condition due to cessation of the AA treatment. The control, SH, SM, and SL mice did not exhibit renal morphological alterations, and data related to biochemical analysis showed no significant differences. Thus, LMWC did not lead to toxicity in the kidneys of C3H/He mice. Among the AA-treated groups, the AM group had the lowest urinary microalbumin and urinary-NAG levels. The AM group recov-

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ered from renal damage indicating that oral administration of 500 mg LMWC/kg BW/day may be the optimum dosage for curing renal lesions in C3H/He mice. Renal histological examination revealed that three different dosages (1500, 500, and 250 mg LMWC/kg BW/day) effectively reversed the AA-induced renal histopathological alteration, particularly treatment with 500 mg LMWC/kg BW/day. A number of researchers have demonstrated that chitosan binds urea, ammonium, and certain acidic substances. However, these researchers have also reported that chitosan does not bind creatinine in in vitro experiments (Maezaki et al., 1993). Our results indicated that LMWC decreases serum creatinine; therefore, restoring the function of renal tubular cells with LMWC might explain the reduction in serum creatinine levels. Based on pathological findings, we conclude that AA induced nephrotoxicity in tested animals and toxic nephrosis include: (a) acute tubular necrosis; (b) hyaline casts; (c) tubular dilatation; and (d) infiltration. Proximal tubular epithelium necrosis (lacking a nuclei, with intense eosinophilic homogenous cytoplasm, but preserved shape) due to interference of AA on epithelial cell metabolic function characterises acute tubular necrosis. Necrotic cells fall into the tubule lumen, obliterate it, thereby causing acute renal failure. Oral administration of LMWC repairs the renal damage caused by AA. The most effective dose is approximately 500 mg/kg BW for C3H/He mice. This study indicates that AA treatment impairs renal function, and oral administration of LMWC in a suitable dose (500 mg/kg BW for C3H/He mice), improves renal lesions in mice. Low molecular weight chitosans show promise as a beneficial supplement. Acknowledgement The National Science Council, Taiwan (NSC98-2313-B-126-003MY3 and NSC97-2313-B-126-003-MY3) partially supported this research. Its financial support is greatly appreciated. References Attaluri, S., Bonala, R. R., Yang, I. Y., Lukin, M. A., Wen, Y., Grollman, A. P., et al. (2010). DNA adducts of aristolochic acid II: Total synthesis and site-specific mutagenesis studies in mammalian cells. Nucleic Acids Research, 38(1), 339–352. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72, 248–254. Dygert, S., Li, L. H., Florida, D., & Thoma, J. A. (1965). Determination of reducing sugar with improved precision. Analytical Biochemistry, 13, 367–374. Hsu, D. Z., Wana, C. H., Hsu, H. F., Lin, Y. M., & Liu, M. Y. (2008). The prophylactic protective effect of sesamol against ferric–nitrilotriacetate-induced acute renal injury in mice. Food and Chemical Toxicology, 46, 2736–2741. Hung, T. H., Chang, Y. M., Sung, H. Y., & Chang, C. T. (2002). Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain. Journal of Agricultural and Food Chemistry, 50, 4666–4673. Jing, S. B., Li, L., Ji, D., Takiguchi, Y., & Yamaguchi, T. (1997). Effect of chitosans on renal function in patients with chronic renal failure. The Journal of Pharmacy and Pharmacology, 49(7), 721–723. Kaltwasser, H., & Schlegel, H. G. (1966). NADH-dependent coupled enzyme assay for urease and other ammonia-producing systems. Analytical Biochemistry, 16, 132–138. Kendra, D. F., & Hadwiger, C. A. (1984). Characterization of the smallest chitosans oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Experimental Mycology, 8, 276–284.

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