An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors

Abstract

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherinpositive, and ZO-1-positive by immunostaining) and BSN cells, which are largely 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 factor (HGF) and transforming growth factor-␤1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early in vitro branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor ␣, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and͞or maintenance of branching tubular structures with lumens in vitro.In the murine embryo, metanephrogenesis is initiated when the ureteric bud (UB) interacts with undifferentiated metanephric mesenchyme around 11.5 days after conception. The UB undergoes successive dichotomous branching steps as it invades the metanephric mesenchyme, developing into the kidney collecting system and ureteric tree. The cells of the metanephric mesenchyme, which have been induced by the UB, epithelialize and ultimately develop into the more proximal nephron from the glomerular capsule to the distal tubule

Staged in vitro reconstitution and implantation of engineered rat kidney tissue

“…Of note, it has been shown that the UB cell line can form tubules under conditions somewhat similar to those we have shown as optimal for branching of the isolated UB (48). It has also been shown that adult ''progenitorlike'' cells from the injured mouse kidney can form tubular epithelial structures in vitro and migrate to multiple compartments of the developing kidney in organ culture (14).…”

Activation of Hepatocyte Growth Factor (HGF) by Endogenous HGF Activator Is Required for Metanephric Kidney Morphogenesis in Vitro

“…The Met receptor is expressed by metanephric mesenchyme as well as by the epithelium of the ureteric bud and later by S-shaped bodies and tubules. Neutralizing antibodies against HGF blocked branching morphogenesis by the ureteric bud in organ cultures of kidney rudiments and inhibited the early steps in branching morphogenesis by immortalized ureteric bud cells (UB cells) in threedimensional organ culture (7,8). Antibodies against HGF, in addition to their effects on branching morphogenesis, inhibited glomerulogenesis and nephrogenesis.…”

Low Ambient O2Enhances Ureteric Bud Branching In Vitro

“…6,7,12 Nigam and coworkers utilized isolated rat metanephros organ cultures and explants of isolated UBs to show that multiple growth factors including FGFs support UB growth and branching in vitro. 16,17 Qiao et al (this group) showed that addition of soluble FGF receptors or of FGF blocking antibodies to organ cultures inhibits UB branching. 16 Several FGFs including FGF2 in combination with GDNF and BSN-CM regulated isolated UB growth and branching in vitro.…”