Congenital abnormalities of the kidney and urinary tract (CAKUT) account for ~50% of childhood chronic kidney disease cases. Many CAKUT defects involve imprecise sizing or spacing of kidney collecting duct structures, such as duplicated ureters, cystic collecting ducts, and renal hypoplasia, which can lead to severe health problems including end-stage kidney disease. As such, there is a critical need to understand how developmental processes promote proper sizing, spacing, and positioning of kidney structures, so that CAKUT defects can be corrected. Kidney development begins through tree-like outgrowth of the ureteric bud epithelium (the future collecting duct network) into a loose connective tissue or mesenchyme. Precisely positioning ureteric bud tubules within this network requires tight control of Ret kinase signaling. Ret responds to secreted glial cell-derived neurotrophic factor (GDNF) from surrounding mesenchymal cells and mutations that affect Ret-GDNF signaling cause CAKUT defects. While Ret-GDNF signaling drives the proliferative expansion of ureteric bud epithelial cells, emerging evidence suggests that ?planar cell polarity? (PCP) ? a mechanism by which cells sense their planar positions within sheets and tubes ? controls the shape of the collecting duct network. Significantly, recent findings indicate that mesenchymal cells expressing the PCP genes Fat4 and Dchs1 prevent improper ureteric bud branching and mutations in these genes cause CAKUT defects in mice and humans. However, it is unclear how interfaces between PCP-expressing mesenchymal cells and Ret-expressing epithelial cells enforce the precise sizing and spacing of collecting duct tubules and avoid defects. The objective of this proposal is to create controlled spatial interfaces between PCP-expressing cells and Ret-expressing cells and study their impact on Ret-GDNF signaling levels and the resultant effect on the size and shape of epithelial structures.
Aim 1 of this proposal establishes a DNA-based cell patterning technology that enables the production of cell interfaces in engineered tissues. This cell patterning technology will be used to create interfaces between PCP-expressing cells and Ret-expressing cells. Cells expressing fluorescence-based kinase activity reporters and mathematical modeling will be used to study how these spatial interfaces influence Ret-GDNF signaling.
Aim 2 of this proposal will pattern the two cell types in 3D tissue scaffolds and study the effects of interfaces on the size and shape of resulting epithelial structures. The central hypothesis of this proposal is that PCP-expressing cells locally restrict Ret-GDNF-driven epithelial tissue growth at interfaces, thereby producing structures of defined size and shape. Together, the approaches developed in this proposal will improve our understanding of the cellular mechanisms that cause CAKUT defects and create new tools to build defined tissue structures in organoid models of human disease.
The kidney performs a vital role in filtering waste products from the bloodstream and directing urine through a tree-like network of tubules. Biochemical and physical interactions between cells must be tightly coordinated in space and time to build such complex structures and the failure of this accounts for the approximately one third of all birth defects that involve the kidney and urinary tract. This project proposes to use engineered tissues to understand signaling pathways that drive and sculpt kidney tubule network development, enabling us to study the causes of congenital kidney defects and create more life-like engineered models of kidney disease.
