Therefore, the objective of the present study was to compare AAI and OTA impact on VEGF expression as well transcription factors regulating its expression in cultured kidney tubulus cells. Comparison between effects of both toxins on VEGF expression may add to our understanding of the role of these toxins in nephropathy development. Aristolochic acid I (AAI), ochratoxin A (OTA), mithramycin A, thiazolyl blue tetrazolium

bromide (MTT), dichlorofluorescein diacetate (DCFH-DA) and SYBR Green were obtained from Sigma–Aldrich. Oligo(dT) primers, dNTP’s, M-MLV reverse transcriptase, Non-radioactive Cytotoxic Lactate Dehydrogenase (LDH) assay and Luciferase Activity Assay were obtained from Epigenetic pathway inhibitor Promega and chetomin was from Alexis Biochemicals. The ELISA kit for human VEGF was procured from R&D Systems Europe. The cell proliferation BrdU ELISA kit was

bought from Roche, the Great Escape SEAP Chemiluminescent Detection kit was from Clontech BD Biosciences and SuperFect Transfection Reagent was procured from Qiagen. High glucose DMEM medium was from Cytogen. Fetal bovine serum (FBS) and antibiotics (penicillin, streptomycin) were from PAA. Rabbit polyclonal anti-HIF-1α (cat no. sc-10790) and anti-HIF-2α (cat no. sc-28706) antibodies were purchased from Santa Cruz Biotechnology, mouse HIF inhibitor monoclonal anti-α-tubulin (cat no. CP06) was from Calbiochem, anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (cat no. 7074) was from Cell Signaling Technology whereas anti-mouse IgG conjugated with HRP (cat no. 32230) was from Pierce. Goat anti-rabbit IgG conjugated with Alexa Fluor 568 (cat no. A21069) was from Invitrogen. Mounting medium with DAPI was bought from Vector Laboratories. LLC-PK1 cell line, an established epithelial cell line derived from porcine renal cortex, was kindly supplied by Prof. Gerald Rimbach (Institute of Human Nutrition and Food Science, IMP dehydrogenase Christian Albrechts University Kiel, Germany).

The cells were passed in high glucose DMEM medium, supplemented with 10% FBS, streptomycin (100 U/ml) and penicillin (100 g/ml), and kept under standard conditions (37 °C, 5% CO2). Toxins were prepared as a 50 mM stock solution (AAI in DMSO, and OTA in methanol). In experiments with mithramycin A and chetomin, cells were pretreated for 30 min with mithramycin A or with chetomin, and then costimulated with AAI for next 24 h. For hypoxia experiments, cells were treated with OTA and then put into hypoxic conditions (0.5% O2, 5% CO2, 94% N2) for 24 h. The effect of AAI (1–100 μM) and OTA (2.5–100 μM) on porcine kidney cell viability has been determined by non-radioactive cytotoxic LDH assay and MTT conversion according to provider’s instruction. LLC-PK1 cells were seeded at a density of 5,000 cells per well in a 96-well plate. After 24 h non-confluent cells were stimulated by OTA and AAI and then cell proliferation was assessed by bromodeoxyuridine incorporation by the use of BrdU ELISA kit according to manufacturer’s instructions.