Anthocyanin-rich Seoritae extract ameliorates renal lipotoxicity via activation of AMP-activated protein kinase in diabetic mice

Preparation of SE and analysis of anthocyanins in SE

The SE used was produced by the following method. Seoritae samples (1,500 g) were
extracted with 12,000 mL of 30% ethanol for 3 h at 90–100°C. The solution was filtered
twice through 50 and 1-µm filters and concentrated in a vacuum evaporator (60°C) to
70 brix. The residual solvent was removed in a drying machine for 18 h at 60°C in
a vacuum. The resulting powder was stored in a plastic bag until use. The anthocyanin
content in SE was analysed by HPLC using a Dionex Ultimate 3000 series dual low-pressure
ternary gradient pump (Dionex Softron GmbH, Germering, Germany) and an Ultimate 3000
series photodiode array detector for anthocyanin analysis. The crude anthocyanin extract
was analysed by its HPLC chromatogram. Three principal anthocyanin peaks were detected
in the chromatogram by diode array detection at 530 nm. The major anthocyanins were
identified as delphinidin-3-O-glucoside (25.2%), cyanidin-3-O-glucoside (68.3%), and petunidin-3-O-glucoside (6.5%) by comparison with the HPLC retention times of standard compounds,
as previously described 19].

Experimental methods

Male 6-week-old C57BLKS/J db/m and db/db mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The mice were
fed a regular chow diet, provided with water ad libitum, and allowed to acclimatise
for 1 week before experiments. The mice were divided into four groups. Control db/m mice (n = 6) and control db/db mice (n = 6) received drinking water only, while anthocyanin db/m mice (n = 8) and anthocyanin db/db mice (n = 8) received 10 mg/kg body weight anthocyanin-rich SE daily for 12 weeks.
For measurement of 24-h urinary albumin, the mice were placed in individual mouse
metabolic cages (Nalgene, Rochester, NY, USA) every 4 weeks. At week 20, the mice
were anaesthetised by intraperitoneal injection of a mixture of Rompun (10 mg/kg;
Bayer Korea, Ansan, Gyeonggi-Do, Korea) and Zoletil (30 mg/kg; Virbac, Carros, France).
Blood was collected from the left ventricle and centrifuged, and the resulting plasma
was stored at ?70°C for analyses. The kidneys were rapidly dissected and stored in
10% buffered formalin for immunohistochemical analyses. HbA
_1c
was measured from red cell lysates by HPLC (Bio-Rad, Richmond, CA, USA). Triglyceride
and total cholesterol concentrations were determined using an automatic analyser (Model
917; Hitachi, Tokyo, Japan) and commercial kits (Wako, Osaka, Japan). NEFA levels
were measured with a JCA-BM1250 automatic analyser (JEOL, Tokyo, Japan).

Ethics statement

All animal experiments were in accordance with the Laboratory Animals Welfare Act,
Guide for the Care and Use of Laboratory Animals, and Guidelines and Policies for
Rodent Experiments provided by the Institutional Animal Care and Use Committee at
the School of Medicine, The Catholic University of Korea (Approval No. YEO20131601FA).
All procedures complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1996).

Assessment of albuminuria, renal oxidative stress, and intra-renal lipid content

Twenty-four-hour urine collection for measurement of albuminuria was performed using
metabolic cages (Nalgene) at week 20, and urinary albumin concentrations were obtained
by immunoassay (Bayer, Elkhart, IN, USA). To evaluate oxidative stress, we measured
24-h urinary 8-epi-prostaglandin F
_2?
(OxisResearch, Foster City, CA, USA). Intra-renal lipids were extracted using the
method of Bligh and Dyer with slight modifications 20].

Light microscopy study

Kidney samples were fixed in 10% buffered formalin and embedded in paraffin. Histology
was assessed after periodic acid-Schiff staining. The mesangial matrix and glomerular
tuft were quantified for each glomerular cross-section, as previously reported 21]. More than 30 glomeruli, cut through the vascular pole, were counted per kidney,
and the average of the measured areas was used for analysis.

Immunohistochemistry for TGF-?1, type IV collagen, and TUNEL assay

For immunohistochemistry, 4-?m-thick sections were deparaffinised, hydrated in ethanol,
treated with an antigen-unmasking solution containing 10 mmol/L sodium citrate buffer
(pH 6.0), and washed with PBS. The sections were incubated with 3% H
₂
O
₂
in methanol to block endogenous peroxidase activity, and then with 10% normal goat
serum in PBS to block non-specific binding. The sections were incubated overnight
with anti-TGF-?1 (1:100; RD Systems, Minneapolis, MN, USA) and anti-COL IV (1:200;
Biodesign International, Saco, ME, USA) antibodies in a humidified chamber at 4°C.
The bound antibodies were localised with a peroxidase-conjugated secondary antibody
using a Vector Impress Kit (Vector Laboratories, Burlingame, CA, USA) and 3,3-diaminobenzidine
substrate solution. Finally, the sections were dehydrated in ethanol, cleared in xylene,
mounted without counterstaining, and examined in a blinded manner using light microscopy
(Olympus BX-50; Olympus Optical, Tokyo, Japan).

For quantification of proportional areas of staining, ~20 views (×400 magnification)
were randomly located in the renal cortex and corticomedullary junction of each section
and images were taken. The images were analysed to determine the density X-positive
area/glomerular total area using a computer image analysis program (Scion Image Beta
4.0.2; Frederick, MD, USA).

Detection of apoptotic cells in formalin-fixed, paraffin-embedded tissue was performed
by in situ TUNEL using an ApopTag In Situ Apoptosis Detection Kit (Chemicon-Millipore,
Billerica, MA, USA). The TUNEL reaction was assessed in a whole glomerular biopsy
under ×400 magnification.

Western blot analysis

Total protein from renal cortical tissues was extracted with Pro-Prep Protein Extraction
Solution (Intron Biotechnology, Gyeonggi-Do, Korea), following the manufacturer’s
instructions. Protein concentrations were determined using the Bradford reagent (Bio-Rad
Laboratories, Hercules, CA, USA). Western blot analysis was performed to further confirm
the responses using antibodies recognising specific epitopes. Proteins were separated
by SDS-PAGE, transferred to nitrocellulose membranes, and detected with the following
antibodies: anti-phosphorylated (phospho)-Thr
¹⁷²
AMPK (Cell Signaling Technology, Danvers, MA, USA), anti-total AMPK (Cell Signaling
Technology), anti-peroxisome proliferator-activated receptor (PPAR) ? (Abcam, Cambridge,
UK), anti-PPAR?, anti-phospho-ACC (Santa Cruz Biotechnology, Santa Cruz, CA, USA),
anti-total ACC (Santa Cruz Biotechnology), anti-sterol regulatory element-binding
protein 1 (SREBP-1; Santa Cruz Biotechnology), anti-B cell leukaemia/lymphoma 2 (BCL-2;
Santa Cruz Biotechnology), anti-BCL-2-associated X protein (BAX; Santa Cruz Biotechnology),
and anti-?-actin (Sigma-Aldrich, St Louis, MO, USA). After washing, the membranes
were incubated with anti-mouse IgG or anti-rabbit IgG horseradish peroxidase (HRP)-linked
secondary antibodies (Cell Signaling Technology) or a rabbit anti-goat IgG HRP-peroxidase
secondary antibody (Sigma-Aldrich). The membranes were developed using an ECL Plus
Detection Kit (Amersham International, Buckinghamshire, UK) to produce chemiluminescence
signals, which were captured on X-ray films. Band densities were quantified with Quantity
One software (Bio-Rad Laboratories).

Cell culture and small interfering RNA (siRNA) transfection

HGECs were purchased from Anigio-Proteomie (Boston, MA, USA) and subcultured in endo-growth
media (Angio-Proteomie). After treatment with medium containing different concentrations
of D-glucose (5 mmol/L D-glucose (low glucose), 40 mmol/L D-glucose (high glucose), or 5 mmol/L D-glucose plus 35 mmol/L mannitol (osmotic control)) and 1, 10, or 50 µg/mL anthocyanin
for 6 h, western blotting was performed for phospho-Thr
¹⁷²
AMPK, total AMPK, PPAR?, PGC-1?, ERR-1?, PPAR?, phospho-ACC, total ACC, superoxide
dismutase (SOD)-1, SOD-2, BCL-2, BAX, and ?-actin with specific antibodies. To examine
the effects of anthocyanins on other renal cells in high-glucose medium, we also used
cultured NMS2 mesangial cells (see Additional file 1 for further details).

siRNAs targeted toward Ampk?1, Ampk?2, and Sirt1, and a non-specific scrambled siRNA
(siRNA control) were complexed with a transfection reagent (Lipofectamine 2000; Invitrogen,
Carlsbad, CA, USA), according to the manufacturer’s instructions. The sequences of
the siRNAs were: ?1-AMPK, 5?-GCAUAUGCUGCAGGUAGAU-3?; ?2-AMPK, 5?-CGUCAUUGAUGAUGAGGCU-3?;
SIRT1, 5?-CACCUGAGUUGGAUGAUAU-3?; and scrambled siRNA, 5?-CCUACGCCACCAAUUUCGU-3? (Bioneer,
Daejeon, Korea). At 24 h after transfection, HGECs were exposed to 40 mmol/L glucose
and 50 µg/mL anthocyanins for 24 h.

Statistical analysis

Data are expressed as mean ± SD. Differences between groups were examined for statistical
significance by ANOVA with the Bonferroni correction using SPSS version 19.0 (IBM
Corp., Armonk, NY, USA). Values of p  0.05 were considered statistically significant.