Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.

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Hepatocyte growth factor (HGF) induces migration, proliferation, and branching in renal epithelial cells from the inner medullary collecting duct (mIMCD-3 cells). Microarray analysis after HGF stimulation of these cells revealed upregulation of the chemokine KC. We found that both the message and protein levels of KC are increased after HGF treatment and that mIMCD-3 cells express the KC receptor CXCR2. Treatment with KC results in stimulation of mIMCD-3 cell proliferation but has no effect on basal rates of cell migration or branching morphogenesis. In contrast to its known stimulatory effect on neutrophil migration, KC markedly inhibits HGF-mediated cell migration and branching morphogenesis, resulting in shorter tubules with fewer branch points. Examination of the mechanism of this effect reveals that KC does not alter phosphorylation of the c-met receptor or the initial activation of the MAPK or phosphoinositide 3-kinase (PI 3-K) signaling pathways. However, sustained activation of the PI 3-K pathway by HGF was inhibited by treatment with KC, and mimicking this effect by treatment with LY-294002 2 h after HGF stimulation reproduced the inhibition of HGF-stimulated branching morphogenesis. These data demonstrate that HGF-mediated KC production can act in an autocrine fashion to downregulate excessive branching and migration of renal epithelial cells in response to HGF, while still supporting cell proliferation. These characteristics may play a role in modulating the response to HGF during developmental tubule formation and/or during the repair of the tubular architecture following injury.