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 cotransport system, gamma-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 alpha-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.
Taub
Mary
M
Department of Biochemistry, State University of New York at Buffalo, New York 14214, USA. biochtau@buffalo.edu
Han
Ho Jae
HJ
Rajkhowa
Trivikram
T
Allen
Cheryl
C
Park
Jun Hong
JH
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002461:Cell Line, Transformed / Q000166:cytology / Q000378:metabolism*; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006728:Hormones / Q000378:metabolism / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011817:Rabbits; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D006728:Hormones; D009004:Monosaccharide Transport Proteins; D000242:Cyclic AMP; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase