Cloning of the pig PEPT2 (pPEPT2) and characterization of the effects of epidermal growth factor (EGF) on pPEPT2-mediated peptide uptake in the renal porcine cell line LLC-PK1.
The renal di/tri-peptide transporter PEPT2 is situated in the distal parts of the proximal tubule, where it mediates reabsorption of peptides from the primary urine. The transporter has been thoroughly characterised with respect to substrate-affinity relationships, however little is known about its regulation. Previous studies from our group have shown that epidermal growth factor (EGF) down-regulates PepT2 in the rat proximal kidney tubule cell line SKPT0193 cl.2. The aim of the present work was to clone the pig PEPT2 (pPEPT2) and to study the effect of EGF on pPEPT2 expression in the porcine kidney cell line LLC-PK1. pPEPT2 from LLC-PK1 cells was PCR-cloned. The predicted protein consisted of 729 amino acids, had a molecular mass of 81.7 kDa and was 88% identical and 94% similar to hPEPT2, thus displaying a close similarity to the human orthologue. pPepT2 expressing LLC-PK1 cells were cultured in the absence and presence of EGF in the culture media. EGF induced an increase in uptake of (14)C-glycylsarcosine ([(14)C]-Gly-Sar), accompanied by an increase in transcellular electrical resistance, total cell protein, alkaline phosphatase activity and cell density. The increase in uptake of [(14)C]-Gly-Sar was maximal when cells were cultured in the presence of EGF throughout the culture period of 10 days. The EGF-treatment did not induce significant changes in pPepT2 mRNA expression, as determined by real-time PCR. The effect of EGF thus appears to be an increase in the number of cells without a loss of differentiation, an effect which is quite different from earlier observations on the SKPT cell line.
Søndergaard
Helle Bach
HB
Molecular Biopharmaceutics Group, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Bravo
Silvina A
SA
Nielsen
Carsten Uhd
CU
Frokjaer
Sven
S
Brodin
Birger
B
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D003470:Culture Media; D004151:Dipeptides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D013552:Swine; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology
D003470:Culture Media; D004151:Dipeptides; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine; D004815:Epidermal Growth Factor; D000469:Alkaline Phosphatase