Non-fermented CSVA were obtained from CSVA in Gimhae, Korea. Twenty grams of CSVA leaves were extracted with 400 mL of 100% methanol in a round-bottom flask equipped with a condenser. The temperature of extraction was kept at boiling temperature and the extraction mixture was constantly stirred with a magnetic bar. After two hours of extraction, the extraction mixture was cooled, the vacuum filtered (0.45 µm), and the methanol evaporated under the vacuum at 40℃.

MDCK cells (American Type Culture Collection, Washington D.C, USA) were cultured in a Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37℃, in air containing 5% CO₂.

MDCK cells were subcultured in 96-well plates and grown to 70-80% confluence. The cells were then exposed to 1 mM of sodium oxalate (Alfa Aesar, Ward Hill, MA, USA) to induce renal cell damage and various concentrations of CSVA extract (0, 10, and 25 µg/L) for 24 hours. Subsequently MTT (0.1 mg, 50 µL of 2 mg/mL) in phosphate-buffered saline was added, followed by incubation for four hours at 37℃. Optical densities were measured on a microplate reader (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 540 nm.

MDCK cells were exposed to 1 mM of sodium oxalate for various periods of time (30, 60, and 120 minutes) with or without 50 µg/L of CSVA extract. The lipid peroxidation levels were then evaluated by measuring the MDA as described by Wong, et al.8

All surgical and experimental procedures were approved by the Seoul National University Institutional Animal Care and Use Committee (SNUIACUC, No. 08-0213) and conducted between November 2008 and February 2009. Thirty male C57BL/Ks db/db mice were divided into three groups by random sampling methods after adaptation to their new environment for one week. Group 1 (n=10) were fed AIN-93G ad libitum. Group 2 (n=10) were fed AIN-93G mixed with 10% fermented CSVA ad libitum. Group 3 (n=10) were fed AIN-93G mixed with 10% non-fermented CSVA ad libitum. Table 1 shows the composition of the basal and experimental diets in each group.

We monitored the total amounts of food and water intake and the body weight of the mice every week. At 6 and 12 weeks of oral intake, blood was collected from the tail-vein and fasting blood glucose levels were measured using Accu-Chek Performa blood glucose monitoring system (glucometer) (Roche Diagnostics, Pleasanton, CA, USA). After 14 weeks of the designated oral intake, 24-hour urine was collected from each mouse in their metabolic cages. We evaluated several factors to assess the effects of CSVA on diabetic nephropathy. These factors included the results of the 24-hour urine analysis, which measured creatinine clearance (Ccr), amount of microalbumin, and total protein. In addition, we examined the histology of the kidneys of all experimental mice to determine the effects of CSVA on the morphology of diabetic nephropathy. For these examinations, all mice were sacrificed using the cardiac puncture technique after fasting for 12 hours. The kidneys were carefully removed after the abdominal cavity was opened. The fatty tissue was gently removed from the renal parenchyma and the kidneys were weighed. For the histological examination under an electron microscope, we excised 1×1 mm of cortical tissue from each kidney and immediately fixed the samples in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4℃ for 4 hours and wash them twice (each time for 1 hour) with 0.1 M buffer. Then, we performed secondary fixation in 2% osmium tetroxide in 0.2 M buffer for 1 hour and rinsed twice (each time for 5 min) in 0.2 M buffer and multistep dehydration in 70%, 90% and 100% ethanol. After propylene oxide infiltration, epoxy resin was treated for overnight and embedded with freshly prepared resin. Finally, the specimens were polymerized at 60℃ for 48 hours. Following the E-firm block formation, ultrathin sections (60 nm) were obtained and subjected to electron staining, meaning double staining with uranyl acetate for 30 min and lead citrate for 10 min; the specimens were then viewed on transmission electron microscopy (JEM-1200EX, JEOL, Tokyo, Japan) operating at 120 kV, which was documented by the digital imaging. The remnant renal tissues were then placed in formalin for paraffin block formation. The paraffin blocks were sectioned and then stained with hematoxylin and eosin (H&E). The tissues were then carefully examined by Olympus SZX7 stereomicroscope (Olympus, Tokyo, Japan) with a plain objective.

The Statistical Package for the Social Sciences (SPSS), version 11.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. All experimental results were analyzed using ANOVA and Dunnett's test and are reported as mean values±standard deviation. p<0.05 was accepted as statistically significant.