UC San Diego

The initiating step in metanephric kidney development is outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) in response to signals arising from the metanephric mesenchyme. Defects in this critical morphogenetic process have been linked to some of the most common birth defects in humans. Despite its obvious importance in kidney development, much remains to be elucidated about the molecular regulation of the process. In order to address this problem, an in vitro model system was utilized which allows for direct assaying of the effects of positive and negative regulators of budding. Study of this regulation can lead to better understanding of the budding process, which in turn can lead to improved treatment or to refined tissue engineering approaches. Although a wealth of literature exists to implicate the glial cell-line derived neurotrophic factor (GDNF) Ret pathway in UB budding, the precise signaling pathways activated by Ret have not been thoroughly studied. Of the various pathways examined, the PI3-kinase/Akt signaling pathway was found to be essential for budding while other Ret stimulated pathways appeared to be dispensable for UB budding. In addition, microarray analysis of numerous budded and unbudded conditions revealed a cluster of genes related to GDNF-Ret signaling involved in budding. Among the identified genes, neuropeptide Y (NPY) was the highest scoring gene product and correlated most significantly to the budded condition out of over 28,000 genes present on the cDNA microarray chip. Although NPY has not been previously implicated in kidney development, it has been shown to be involved in the GDNF-dependent development of enteric neurons, thus it is an ideal candidate for modulation of GDNF-dependent budding from the Wolffian duct. When NPY was added to the cultured Wolffian duct with GDNF impressive budding was observed; conversely, inhibition of the NPY receptors inhibited budding, confirming that NPY facilitates this process. Addition of BMP4 decreased budding through either downregulation of Ret and GFR[alpha]1 or by blocking the PI3-kinase/Akt signaling pathway. This may explain how endogenous BMP4 inhibits ectopic budding. Addition of NPY to these BMP4-treated WDs rescued budding with a corresponding increase in phosphorylated Akt as well as Ret and GFR[alpha]1 expression. This suggests that NPY may act through this pathway. This represents a novel mechanism for NPY in the development of the kidney. The formation of the ureteric bud may be a result from a combination of upregulation of the GDNF receptors along with genes that support GDNF signaling in a feed-forward loop. Experiments performed with WDs lacking the attached intermediate mesodermal cells suggest that there is an element produced by these mesenchymal cells that augments the budding processes