Low-molecular-weight chitosan scavenges methylglyoxal and N ? -(carboxyethyl)lysine, the major factors contributing to the pathogenesis of nephropathy

Chemicals

Crab shell chitosan (84% deacetylated; approximately 1,100 kDa) was obtained from
Ohka Enterprises Co. Ltd. (Kaohsiung, Taiwan). A CEL detection kit was obtained from
Cell Biolabs Inc. (San Diego, CA, USA). A glutathione assay kit was obtained from
Cayman Chemical Co. (Ann Arbor, MI, USA). Anti-CEL antibodies were purchased from
Cosmo Bio Co. (Tokyo, Japan). A polymer detection system, which includes blocking
reagent A and B, was purchased from Nichirei Biosciences Inc. (Tokyo, Japan).

Preparation of chitosanase and lmw-chitosan

Chitosan was used to induce bamboo chitosanase, which was in turn utilized to digest
chitosan (1,100 kDa) into lmw-chitosan (~29 kDa), as previously described (Chang et
al. 2011]). Briefly, bamboo (Bambusa oldhamii) shoots were coated with chitosan and stored at 25°C to induce chitosanase, which
was isolated, and its activity was measured. Chitosan was suspended in 4.5% acetic
acid, digested with 180 U chitosanase at 50°C for 18 h, and purified before use.

Determination of the inhibitory effects of lmw-chitosan on MG in vitro and in kidney
samples

MG levels were determined using HPLC. Lmw-chitosan was diluted to different concentrations
(1, 2, 5, 10, and 40 µg mL
^?1
) for testing the MG inhibition effect in vitro. In addition, kidney homogenates were
diluted fourfold, and 20 ?L diluted homogenate was used to detect the MG level. To
each sample, 2 ?L ammonium chloride buffer (pH 10) was added to create an alkaline
environment for the derivatization reaction. The derivative reagent, 50 µL 7.5 × 10
^?4
 M DDP, was added to each sample, and the reaction mixture was incubated at 60°C for
30 min in the dark. The reaction was then stopped by addition of 500 µL 0.01 M citric
acid buffer (pH 6). The mixture was vortexed, spun down, incubated on ice for 10 min,
and then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was filtered
with a 0.45 µm filter to remove impurities and then layered onto an ODS column for
separation at 33°C (Biosil, 250 mm × 4.6 mm ID; 5 ?m particle size; Biosil Chemical
Co. Ltd., Taipei, Taiwan). Acetonitrile was used as the mobile phase: 0.01 M citric
acid buffer = 3:97, v/v; flow rate = 0.7 mL/min, Ex/Em = 330/500 nm. All samples were
analyzed within 24 h.

Animal treatment and sample preparation

A total of 23 six-week-old female C3H/He mice were purchased from the National Laboratory
Animal Breeding and Research Center (Taipei, Taiwan). The mice were divided into three
groups of six mice each plus a control group consisting of five animals after 1 week
of acclimation. The control (Group C) and lmw-chitosan only (Group M) groups were
injected with 0.1 mL normal saline each day for 5 days (days 1–5, IP), whereas the
disease (Group A) and therapy (Group AM) groups were injected with 0.1 mL 10 mg kg
^?1
 AA (IP) each day for 5 days. The mice in Groups AM and M then received lmw-chitosan
(500 mg kg
^?1
 day
^?1
, PO) for 14 days after AA injection, while mice in Groups C and A received the same
volume of water. The mice were anesthetized with isoflurane and sacrificed by drawing
blood from the inferior vena cava. The kidneys were then harvested and immediately
put on ice. The kidneys were homogenized with a motorized homogenizer unit at 6,000 rpm
by using phosphate buffered saline (PBS); the resulting homogenates were stored at
?80°C until use.

Determination of CEL concentrations in kidney samples

The CEL concentrations were determined using a commercial kit. Briefly, all kidney
homogenate samples were diluted in PBS to a final total protein content of 10 ?g mL
^?1
. One hundred microliters of each sample or standard was then added to a protein adsorbent
plate, which was incubated overnight at 4°C. After incubation, the plate was washed
with PBS twice and subsequently incubated with 200 µL Assay Diluent buffer for 1.5 h.
The plate was then washed three times with 250 ?L wash buffer and incubated with anti-CEL
antibodies at room temperature for 1 h on an orbital shaker. Then the plate was again
washed with wash buffer three times and incubated with secondary horseradish peroxidase-conjugated
antibodies for 1.5 h at room temperature on an orbital shaker. Next, the plate was
washed with wash buffer five times and then incubated with 100 ?L Substrate Solution
at room temperature for 15 min on an orbital shaker. The enzyme reaction was stopped
by adding 100 ?L Stop Solution to each well. The absorbances of the reaction mixtures
were read immediately on a microplate reader at 450 nm. Reduced bovine serum albumin
was used as an assay blank.

Immunochemical staining of CEL

To determine the localization of CEL by immunohistochemistry, mouse kidneys were fixed
with 4% paraformaldehyde for 2 days, dehydrated with different concentrations of ethanol
and xylene, embedded in paraffin, and sectioned to 5 ?m thickness. After blocking
endogenous peroxidase activity by treating the sections with 3% H
₂
O
₂
for 10 min, slides were washed three times with PBS. The slides were then incubated
with blocking reagent A for 60 min at room temperature. Anti-CEL antibodies were diluted
50-fold and incubated with the samples overnight, followed by washing with PBS. Next,
the samples were incubated with blocking reagent B for 10 min at room temperature.
The samples were then incubated with a secondary antibody for 10 min at room temperature.
Subsequently, the tissue sections were washed for a final three times and then dried
carefully. Two drops of the chromogen/substrate reagent was added to each section,
and the samples were incubated in the dark at room temperature for 10 min. Sections
were then counterstained with hematoxylin. Staining without inclusion of the primary
antibody was performed as the negative control.

Determination of glutathione concentrations in kidney samples

The glutathione levels in the kidney samples were measured using a glutathione assay
kit (Cayman), as previously described (Li et al. 2012]). Briefly, the tissue homogenates were added to a cocktail buffer containing glutathione
reductase, glucose-6-phosphate dehydrogenase, and 5,5?-dithiobis-(2-nitrobenzoic acid)
(DTNB). The mixture was incubated at 37°C in the dark, and the absorbance was measured
at 405 nm at 5-min intervals over 30 min.

Statistical analysis

All data were expressed as means ± standard errors of the means (SEMs, where n ? 5).
The differences were analyzed using one-way analysis of variance (ANOVA), and the
levels of significance among various treatments were determined using Scheffe’s multiple
range test. Differences with p values  0.05 were considered statistically significant.