Study On The Renal Protective Effects Of Nuclear Factor-κB Decoy Oligodeoxynucletides In Ischemic Acute Renal Failure Model

PART 1Transfection of Nuclear factor-κB decoy oligodeoxynucleotides inhibits inflammatory genes expression in renal tubular epithelial cellsBACKGROUNDThe pathophysiology of ischemic acute renal failure is complex. Endothelins, adhesions, chemokines and inducible nitric oxide synthase (iNOS) might be involved in ischemic renal injury. Because these genes are targets for NF-κB, the use NF-κB decoy oligodeoxynucleotides (ODN) strategy mediated by protamine liposome was evaluated in NRK-52E renal tubular epithelial cells in vitro.METHODSLipsome (1﹕1 DOTAP/cholesterol) and ODNs complexes were prepared at +/-charge ratio of 4 and then transfected into TNF( stimulated, complete cultured (contained 20% fetal bovine serum) NRK-52E cells. The transfection efficiency was observed by fluorescence microscope and evaluated by flow cytometry. NF-κB DNA binding abilities were analyzed by electrophoretic mobility shift assay (EMSA). Genes expression of endothelin-1 (ET-1), iNOS, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecular-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were assessed by reverse transcription polymerase chain reaction (RT-PCR).RESULTSCompared to naked transfection, mediated by protamine liposome produced high transfection efficiency. The efficiency was 93.20% and 39.0%, respectively, 2 hours after transfection. FITC-labeled ODN fluorescence was mainly localized in nuclei. NF-κB DNA binding abilities activated by TNF( were elevated markedly in 30 min, reached to climax in 6h and maintained in the highlevel. It was almost abolished by NF-κB decoy ODN, instead of scrambled ODN. TNF( induced gene expression of ET-1, iNOS, ICAM-1, VCAM-1 and MCP-1 were increased accordingly to NF-κB activities and significantly inhibited by NF-κB decoy ODN ( Ps<0.01 ). CONCLUSIONSProtamine liposome mediated ODN transfection method was highly efficient in vitro and did not affected by serum. Transfection of NF-κB decoy ODN resulted in attenuation NF-κB activities, leading to the inhibition of inflammatory genes expression in TNF( activated renal tubular epithelial cells.