TYROSINE KINASE-ACTIVITY OF THE EGF RECEPTOR IN MURINE METANEPHRIC ORGAN-CULTURE

Epidermal growth factor (EGF) and its fetal form, transforming growth factor alpha (TGF-alpha) are renal mitogens which induce epithelial hy perplasia, proximal tubular cyst formation (TC), and accelerated dista l nephron differentiation in metanephric organ culture. To delineate t he intracellular mechanisms mediating these growth factor effects, we studied the specific role of the epidermal growth factor receptor (EGF -R), the common receptor for both ligands, as an activated tyrosine ki nase in TC formation and nephrogenesis. Fetal murine metanephric expla nts were incubated for 120 hours in control, and EGF (15 ng/ml)/TGF-al pha (10 ng/ml) supplemented medium with and without EGF-R blocking mon oclonal antibody (50 mg/ml), or tyrosine kinase inhibitor. EGF-R tyros ine kinase inhibition was achieved by incubation with a synthetic tyrp hostin (TP B42) (0.1 mu M) or genestein (5.5 mu g/ml). The following p arameters were assessed: (a) segment-specific nephron development usin g morphometry and immunohistology; (b) tubular epithelial hyperplasia by protein content and BrdU uptake; and (c) TC formation by morphometr ic cystic index. Both growth factors produced hyperplastic proximal TC , significantly increased explant growth, and significantly increased distal nephron differentiation. Inhibiting the ligand-EGF-R interactio n with EGF-R blocking monoclonal antibody abolished all growth factor- induced effects and resulted in increased amounts of undifferentiated mesenchyme and decreased distal nephron differentiation. inhibition of EGF-R tyrosine kinase activity with either Tyrphostin B42 or genestei n blocked TC formation and produced nodular blastemal hyperplasia and decreased distal nephron differentiation. Inhibition of EGF-R tyrosine kinase activity may provide a target for pharmacological therapy in h uman cystic disease, in which there are both qualitative and quantitat ive abnormalities of EGF-R expression.