SUMMARY: The distal nephron epithelium acquires essential functions for urinary concentration during its maturation phase following nephrogenesis. We identified KCTD1 as a critical regulator of the maturation and function of the thick ascending limb (TAL) and the distal convoluted tubule (DCT) of the distal nephron. KCTD1 deficiency leads to impaired maturation of these nephron segments with loss of key electrolyte transporters, resulting in an early-onset severe salt-losing tubulopathy and a delayed-onset nephronophthisis-like cyst formation and kidney fibrosis. Mechanistically, lack of KCTD1 increases expression of the epithelial differentiation regulator DAPL1, which we propose suppresses maturation and terminal differentiation of the distal nephron epithelium by increasing β-catenin signaling activity through inhibition of the expression of the Wnt inhibitor SFRP1. Inhibiting the increase in β-catenin activity in mice lacking KCTD1 prevented the loss of electrolyte transporters, improved kidney function and attenuated fibrosis and cyst formation. Thus, KCTD1 controls distal nephron maturation and function by suppressing postnatal β-catenin activation. In this proposal we aim to define the molecular mechanisms through which KCTD1 controls DAPL1 expression and Wnt/b-catenin signaling activity in the distal nephron epithelium and thereby affects the differentiation and function of this epithelium. Our new preliminary data show that increased AP-2a in the TALs/DCTs of mice lacking KCTD1 is required for the increase in Wnt/β-catenin signaling in these TALs/DCTs and that lack of AP- 2a can rescue the distal nephron maturation defects caused by KCTD1 deficiency. Based on these findings we hypothesize that KCTD1 acts in the TALs/DCTs as an inhibitor of the transcription factor AP-2a, which induces the expression of DAPL1. We will test this hypothesis and explore how AP-2a controls DAPL1 expression and whether the functions of KCTD1 for distal nephron maturation are mediated by AP-2a and DAPL1. Moreover, we will test whether DAPL1 stimulates Wnt/b-catenin signaling activity in the TALs/DCTs by inhibiting the expression of SFRP1 and define the molecular regulation and in vivo significance of a KCTD1/AP- 2a/DAPL1/SFRP1 axis in controlling Wnt/β-catenin signaling activity and maturation and function of the distal nephron epithelium. The scientific premise for this application is strong and builds on extensive preliminary data, mouse models that have already been generated and verified in our laboratory, and a primary human TAL/DCT cell culture system for which the feasibility of the proposed experiments has been clearly established by our preliminary data. The proposed experiments are expected to provide fundamental new insights into molecular m...