Autosomal-dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide and is a common cause of end-stage kidney disease (ESKD). ADPKD is caused by mutations in one of two genes, PKD1 or PKD2, which encodes polycystin 1 (PC) and PC2 respectively. Loss of PC1 or PC2 results in activation of numerous kinases and downstream signaling pathways, which is central to the pathogenesis of cyst growth in ADPKD. In addition, pharmacologically inhibiting a number of different kinases up-regulated in PKD kidneys has been shown to slow cyst growth in animal models of PKD making kinase inhibitors among the most promising class of drug candidates to treat patients with ADPKD. However, while the human kinome consists of more than 500 kinases, only a fraction of these kinases have been tested to determine if they play a role in ADPKD pathogenesis. As a result, there are likely many kinases that are active in ADPKD kidneys that play prominent roles in cyst growth that are yet-to-be discovered and may be good therapeutic targets. In collaboration with James Duncan at Temple University, we have now adapted a novel approach to broadly screen PKD kidneys in an unbiased manner for kinases that are more active in lysates from PKD kidneys compared with lysates from wild type kidneys. In this assay, active kinases are affinity captured by passing lysates over multiplex inhibitor beads (MIB) containing a cocktail of kinase inhibitors. Bound kinases are then identified by LC separation followed by quantitative tandem mass spectrometry (LC- MS/MS). Using this approach, we have now identified a number of both known and unknown kinases specifically up-regulated and down-regulated in PKD kidneys. The major goal of this proposal is to assess the role of several of the kinases identified thus far whose activity is increased in PKD kidneys and determine whether inhibiting or activating any of the kinases identified slows cyst growth, inhibits interstitial fibrosis, and preserves renal function in animal models of ADPKD. We will then take both biased and unbiased approaches to identify the signaling pathways regulated by these kinases that are critical to pathogenesis with the goal of developing a more complete picture of the relevant signaling hubs and networks that are aberrantly activated in PKD kidneys. In addition, we will use this technology to broadly screen PKD kidneys from a variety of different “early” and “late” mouse models of ADPKD at different stages in cyst formation and in kidneys from human patients with ADPKD to identify in an unbiased manner additional kinases that are activated and inhibited in PKD kidneys to determine the similarities and differences between mouse models, which kinases may be most relevant to human disease, and whether distinct sets of kinases are activated early post cyst induction and function as early “drivers” of cyst growth. Ultimately, we hope this new information will identify new safe drug targets and rationa...