Renoprotective effect of tectorigenin glycosides isolated from Iris spuria L. (Zeal) against hyperoxaluria and hyperglycemia in NRK-49Fcells

Oxidative stress has been identified as an underlying factor in the development of insulin resistance, β-cell dysfunction, impaired glucose tolerance and type 2 diabetes mellitus and it also plays a major role in kidney stone formation. The aim of the present study is to elucidate the in vitro nephroprotective activity of two isoflavonoid glycosides, tectorigenin 7- O- β-glucosyl-(1→6) β-D-glucoside ( 1 ) and tectorigenin 7- O -β-D-glucosyl-4'- O -β-D-glucoside ( 2 ) isolated from the n -BuOH fraction of Iris spuria L. (Zeal) rhizome MeOH extract against oxalate and high glucose-induced oxidative stress in NRK-49F cells. The results revealed that compounds 1 and 2 significantly increased the antioxidant enzyme activities and decreased MDA levels in both oxalate and high glucose stress. Treatment with these phytochemicals effectively down-regulated expression of crystal modulator genes and pro-fibrotic genes in oxalate and high glucose-mediated stress respectively. The present study indicates cytoprotective, antioxidant, anti-urolithic and anti-diabetic effects of compounds 1 and 2 from I. spuria against oxalate and high glucose stress.


Experimental Section
The compounds were isolated and identified before as described by Singab (2004). The data are also listed here.

Extraction and isolation
The fresh I. spuria rhizomes (1 kg) were cut into small pieces, and subjected to successive extraction with petroleum ether, CHCl 3 , and 70% aqueous MeOH at room temperature. Extracts were concentrated in vacuo at low temperature (45°C). The 70% MeOH extract (45 g) was further extracted with n-BuOH and subsequently evaporated at 55°C concentrated to yield 12 g of a yellowish brown residue. Eight grams of the n-BuOH extract were chromatographed over Sephadex LH-20 eluted with MeOH: H 2 O (9:1) to afford 10 fractions. Fractions 6 and 7 (850 mg) showed two major spots on RP-C18 TLC (RP-C18) using MeOH: H 2 O (1:1) as a solvent system. Separation of the two compounds was carried out by preparative HPLC (Gilson-305 pump equipped with JASCO UV-970 detector and YMC-PACK R&D preparative column (10 µm ODS 120 A, 250x20 mm) using MeOH: H 2 O (1:1) isocratic, and pooling the similar fractions by comparing their TLC profile using an RP-C18 TLC and a solvent system MeOH: H 2 O (1:1) to afford compounds 1 and 2, which were further purified over a Sephadex LH-20 column eluted with 10% aqueous MeOH to yield compounds 1 (65 mg) and 2 (100 mg) Singab (2004). Sephadex column chromatography was used for purification to increase the purity. A column filled with Sephadex LH-20 column (20x 1 cm) was used, the solvent used was 10% aqueous MeOH (90 water: 10 HPLC MeOH).

Cell lines and chemicals
NRK-49F cell line was procured from NCCS (Pune, India), and maintained in the tissue culture facility of the Department of Biochemistry, Madurai Kamaraj University, Madurai, India.
Cells were grown in DMEM supplemented with heat-inactivated FBS (10%), penicillin G (100 IU/ml) and streptomycin (100 mg/ml) and maintained at 37ºC in a 5% CO 2 atmosphere with 95% humidity. Cell culture media and consumables were procured from HiMedia Laboratories Pvt.,

Measurement of intracellular ROS:
Level of net intracellular ROS production was detected by staining NRK-49F cells with 2,7-dichlorodihydrofluorescein diacetate (H 2 DCFDA), a fluorescent probe and visualized using Nikon Eclipse Ti fluorescence microscope (Nikon, Tokyo, Japan).

Antioxidant enzyme profile and lipid peroxidation assay
The antioxidant enzyme activities and lipid peroxidation was measured using cell lysates. In brief, cells were added with ice-cold lysis buffer (Tris-Cl and sodium fluoride, Tris-Cl, 50 mM; NaF, 10mM; NaCl, 0.15 M; EDTA, 2 mM; sodium pyruvate, 1 mM; PMSF, 10 μg/ml; and triton-X, 0.1%) and centrifuged at 5,000 rpm for 10 min. The supernatant was subject to the estimation of protein content, evaluation of antioxidant enzyme activities and lipid peroxidation levels.

Gene expression analysis
Total tissue RNA was extracted using Tri reagent (Sigma-Aldrich Inc. USA) and quantified by Nanodrop Spectrophotometer. RNA samples were reverse transcribed using the MMLV reverse transcriptase (Promega, Madison, WI USA) and oligo-dT primers. The mRNA expression levels of β-Actin, OPN, renin and ACE genes were performed for cells treated with oxalate and flavonoids 1 and 2 by semi-quantitative RT-PCR. Similarly, the gene expression profile of TGF-β1 and fibronectin were carried out in cells exposed to high glucose stress and treatment with flavonoids 1 and 2.

Statistical analysis
Data are expressed as mean ± SEM values, and statistical significance was assessed by one-way ANOVA. Differences were considered to be significant at p < 0.05. (e) NRK49F cells treated with KOx and compound 2 for 18 h.

Figure S3
Effect of isoflavonoids (1 and 2) on cell morphology in hyperglycemic stress.
(a)NRK-49F cells devoid of stress (b) NRK-49F cells exposed to high glucose 30mM for 48h.   Tables   Table S1 Effect of compounds (1 and 2) on antioxidant enzyme activity and lipid peroxidation assay in NRK-49F cells exposed to oxalate (1500μM) Values are given as mean ± SEM from three independent experiments. a Significantly different from the control cells ( P<0.05). b Significantly different from the cells exposed to oxalate ( P<0.05). c Significantly different from the cells exposed to both oxalate and NAC ( P<0.05).