Clonal analysis of immortalized renal proximal tubule cells: Na⁺/glucose cotransport system levels are maintained despite a decline in transport function

Primary rabbit kidney proximal tubule (RPT) cells (S.D. Chung et al., 1982, J. Cell Biol. 95, 118-126) were transfected with the vector pRSV-T, which contains SV40 early region genes. After the third passage (when normal cells had stopped dividing), individual colonies formed in cultures transfected with pRSV-T. Clonal isolates (RPT-I cells) could be obtained in a simple and reproducible manner. Southern analysis of clone RPT-I8 indicated the presence of SV40 early region genes. Nuclear SV40 T was detected. After 23 passages, and subcloning, RPT-I8 (and subclones) was found to express renal proximal tubule markers to a similar extent, indicating that the phenotype was stable. Nevertheless, the activities of the Na⁺/glucose co-transport system, γ-glutamyl transpeptidase and alkaline phosphatase, were reduced as compared with primary cultures. Western analysis indicated that the level of Na⁺/glucose cotransporters was maintained in RPT-I8 cells, when compared with intact proximal tubules and primary cultures. Thus, the reduction in α-MG uptake in RPT-I8 cells may be attributed to other types of cellular alterations, including changes in energy metabolism. Indeed, growth in glucose-free medium was not observed in RPT-I8 cell cultures, suggesting that unlike primary RPT cells (J. C. Chung et al., 1992, J. Cell. Physiol. 150, 243-250), the gluconeogenic pathway was not intact.