CONSECUTIVE USE OF HORMONALLY DEFINED SERUM-FREE MEDIA TO ESTABLISH HIGHLY DIFFERENTIATED HUMAN RENAL PROXIMAL TUBULE CELLS IN PRIMARY CULTURE

Highly differentiated human proximal tubule (HPT) cells in primary cul ture were established from heterogeneous suspension of tubules prepare d from the human renal cortex by an original two-step procedure. First , gluconeogenic-competent HPT cells were selected by using a hormonall y defined serum-free medium without glucose or insulin; then, the sele cted HPT cells were grown in a medium containing a low concentration o f glucose (1 mM) and insulin (0.5 mu g/mL) but no antibiotics. HPT cel ls grown on plastic support formed confluent, cobblestone-like monolay ers with numerous mitochondria and pinocytosis vacuoles, solitary cili a, junctional complexes, and a well-developed brush border consisting of densely packed microvilli, Corrlpared with cell monolayers on plast ic support, HPT cells grown on porous filter membranes showed better m orphologic differentiation. HPT cell monolayers expressed the followin g differentiated functions of the proximal tubule in situ: a tow-affin ity, high-capacity Nat-dependent glucose transport system inhibited by phlorizin, a high-affinity Naf-dependent phosphate transport system, a basolateral organic cation uptake inhibited by mepiperphenidol, para thyroid hormone-sensitive cAMP synthesis, brush-border hydrolase activ ities, gluconeogenesis-associated enzymes, glutathione-S-transferases and N-acetyl-beta-D-glucosaminidase. The medium containing low glucose and insulin concentrations markedly limited the increase in glycolysi s but did not prevent the falls in gluconeogenesis and brush-border hy drolase activity at any time of the culture period. Similar decreases of brush border enzyme activities were obtained for HPT cells grown ei ther on plastic or on porous filter membrane. A thorough characterizat ion study demonstrated that this simple and preparative experimental a pproach makes it possible to establish highly differentiated HPT cells in primary culture suitable for investigating human renal proximal tu bular cell function.