Congenital anomalies of the kidney and urinary tract (CAKUT) constitutes 20-30% of anomalies identified in the prenatal period, and is estimated to play a causative role in up to 50% of cases of end-stage kidney disease in children. CAKUT, affecting approximately 600,000 Americans, encompases a large group of disorders that result from abnormal renal developmental processes. Planar cell polarity (PCP) is the coordinated orientation of cells and cellular structures along an axis within the plane of an epithelial surfac. PCP plays a central role during renal development when newly formed tubules undergo progressive lengthening by directed cell migration and cell division. Wnt family proteins have been shown to control cell polarity and directional cell movement in many developmental systems. The mechanisms by which Wnt signaling pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are not completely understood. The goal here is to understand the mechanism by which Wnt5a integrates downstream organelles and cytoskeletal proteins, as well as PCP proteins, to confer cell polarity and directional cell movement during kidney development. Wnt5a is a noncanonical Wnt that binds to its receptor Frizzled, acts through disheveled (Dsh), and activates both the Wnt/Ca2+ and PCP pathways. I previously showed that Wnt5a nul mice develop defects in prostate gland lumenization and planar cell polarity, and Wnt5a null mice, which die at birth, lack kidneys. Wnt5a appears to be expressed in the mesenchyme/stroma of the developing mouse kidney. The hypothesis, therefore, that we propose testing is that Wnt5a regulates kidney development via actions on the PCP pathway. Accordingly, in Aim 1, I will use in vitro cel culture to investigate the detailed mechanism by which Wnt5a regulates PCP.
In Aim 2, I will determine the in vivo role of Wnt5a signaling for guiding pronephric kidney development, using antisense Wnt5a morpholinos in zebrafish. Use of zebrafish will allow me to rapidly confirm and expand on the mechanistic in vitro results from Aim 1. Finally, in Aim 3, I will determine the role of Wnt5a in controlling PCP and regulating metanephric kidney development in vivo by utilizing the Cre/loxP binary mouse system to generate kidney-specific Wnt5a knockout mice. Wnt5a floxed mice are available to us and I plan to do a whole expression profile of Wnt5a in the developing mouse kidney to confirm reports of Wnt5a expression in developing kidney mesenchyme/stroma. Mesenchymal kidney Cre drivers, such as FoxD1, are commercially available, and I can knockout Wnt5a in both a kidney-specific and temporal manner by mating Wnt5a floxed mice with FoxD1-CreERT2 mice. I will evaluate oriented cell division and other PCP parameters in the Wnt5a knockout model and then investigate downstream elements including PCP proteins. Successful completion of these experiments will result in the identification of novel candidate targets for therapeutic intervention in CAKUT, a disorder for which no approved treatments currently exist.
Congenital anomalies of the kidney and urinary tract (CAKUT) constitute 20-30% of all birth defects identified in newborns, and is the cause of up to 50% of childhood end-stage kidney disease. Wnt5a is a member of the Wnt gene family, a group of very important developmental genes, and mice without Wnt5a lack kidneys. The proposed experiments are designed to determine the mechanism by which Wnt5a regulates kidney development, with the successful completion of these experiments resulting in the identification of novel candidate targets for therapeutic intervention in CAKUT, a disorder for which no treatments currently exist.
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