AN IN-VITRO TUBULOGENESIS SYSTEM USING CELL-LINES DERIVED FROM THE EMBRYONIC KIDNEY SHOWS DEPENDENCE ON MULTIPLE SOLUBLE GROWTH-FACTORS

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development, Two immortali zed cell lines derived from the day 11.5 embryonic kidney, UB cells, w hich appear to be epithelial (cytokeratin-positive, E-cadherin-positiv e, and ZO-1-positive by immunostaining) and BSN cells, which are large ly mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an in vitro tubulogenesis system, BSN cells expressed hepatocyte growth f actor (HGF) and transforming growth factor-beta 1 mRNAs, and its condi tioned medium (BSN-CM) contained factors capable of activating the epi dermal growth factor (EGF) receptor (EGFR), When UB cells were culture d in an extracellular matrix gel in the presence of the embryonic kidn ey or BSN-CM, the UB cells underwent morphogenetic changes characteris tic of early ipl vitro branching tubulogenesis, These changes were lar gely inhibited by a combination of neutralizing anti-HGF antibodies an d the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activi ty, Nevertheless, there was a significant fraction of tubulogenic acti vity that could not be inhibited, suggesting the existence of other so luble factors, Whereas HGF, EGF, transforming growth factor alpha, bas ic fibroblast growth factor (bFGF), and insulin-like growth factor 1 ( IGF-1), or a mixture of these growth factors, induced epithelial proce sses for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly t o the same degree as BSN-CM, Moreover, only BSN-CM induced branching t ubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens in vitro.