Construction of an Erythropoietin-Expressing Bioartificial Renal Tubule Assist Device

Abstract

Methods: A eukaryotic expression vector pcDNA3.1-hEpo was constructed by standard cloning methods. G418 was applied to select for a stable erythropoietin (Epo) expression cell clone. Cell viability and cell growth curve were 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 were detected by enzyme-linked immunosorbent assay (ELISA). The inulin leak rate and the transport of Na+ and glucose (Glu) of the Epo-expressing bioartificial renal tubule assist device (RAD) were determined and compared with the conventional RAD. The specific inhibition of the transport by ouabain and phlorizin was 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. 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 intra- and extraluminal culture media in the novel RAD. There was no significant difference about the inulin leak rate and the transport rate of Na+ and Glu between the novel and conventional RADs (p > 0.05 for each). And the transport function of the novel and conventional RADs was significantly inhibited by ouabain and phlorizin (p < 0.01 for each). 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.