Construction Of The Expression Of Erythropoietin Epo Bioartificial Renal Tubule

Objective To construct a novel bioartificial renal tubule assist device (RAD) which can express erythropoietin (Epo), and explore whether other functions of the RAD were affected after the expression of Epo. Methods A eukaryotic expression vector pcDNA3.1-hEpo was constructed by standard cloning methods. G418 was applied to select for a stable Epo expression cell clone. Cell viability was evaluated and a cell growth curve was generated with MTT chromatometry. HK-2 cells were transfected with pcDNA3.1- hEpo, cultured and then seeded into the hollow fibers of high-flux hemofilters. The expression levels of Epo in the culture supernatants, intraluminal and extraluminal space of the hemofilters were detected by Enzyme-linked immunosorbent assay (ELISA). The transport of Na+ and glucose (Glu) of the Epo-expressing RAD was determined and compared with the conventional RAD, which were seeded with nontransfected HK-2 cells. The specific inhibition of the transport by ouabain and phlorizin was also determined. Results The Epo expression plasmid pcDNA3.1-hEpo was successfully constructed and generated. Stable expression of Epo was obtained by selection of G418 after HK-2 cells were transfected with pcDNA3.1-hEpo. There was no obvious difference about cell growth curves between the transfected and untransfected HK-2 cells (P>0.05). High levels of Epo expression were observed in both intraluminal and extraluminal culture media in the novel RAD. The levels of Epo in the extraluminal space were about 37.20% (24h), 42.42% (48h), 51.25% (96h), and 45.48% (120h) respectively of that in the intraluminal space. There was no significant difference about the transport rate of Na+ and Glu between the novel RAD and the conventional RAD (P>0.05 for each). The transport function was significantly inhibited by ouabain and phlorizin (P<0.01 for each), and recovered after the removal of ouabain and phlorizin in both groups .Conclusions A novel RAD that can express Epo was successfully constructed in vitro, in which other functions of the RAD were not affected by the transfection of pcDNA3.1-hEpo.