Causative mutations of cystic kidney disease occur in genes encoding proteins that localize to primary cilia, yet the molecular events connecting cilia dysfunction to renal cystogenesis remain unclear. In particular, Hedgehog (Hh) signaling, which is affected by all primary cilia structural defects during development, is understudied in renal cystogenesis. The ciliary gene, Thm1 (Ttc21b), negatively regulates Hh signaling and is most commonly mutated in human ciliopathies, which manifest renal cysts as a major clinical feature. Thm1 deletion in a conditional knock-out (cko) mouse causes cystic kidney disease with increased expression of Gli genes, which encode the final mediators of the Hh pathway. Loss of Gli2, the primary transcriptional activator of the Hh pathway, results in less severe cystic kidney disease in Thm1;Gli2 double conditional knock-out (dko) mice supporting a causative role for increased Hh signaling in Thm1 renal cystogenesis. Importantly, Gli genes are also upregulated in established mouse models of Autosomal Dominant Polycystic Kidney Disease (ADPKD), jck and Pkd1, and Hh inhibitors reduced cystogenic potential of jck and Pkd1 cultured kidneys. Thus, enhanced Hh activity may have a general role in renal cystogenesis and may present a valuable target in the treatment of ADPKD. To test this hypothesis, three specific aims will be used. In the first aim, Hh signaling will be inhibited genetically and pharmacologically in developmental and adult-onset mouse models of ADPKD to establish that increased Hh signaling leads to Pkd-mediated renal cysts in vivo. In the second aim, the renal transcriptomes of Thm1 cko and Thm1;Gli2 dko mice prior to and during disease progression will be examined using genome-wide and candidate approaches to determine the molecular mechanism by which increased Hh signaling contributes to renal cystogenesis. Lastly, in the third aim, investigations of Hh signaling will extend to human ADPKD tissue to verify that signaling pathways delineated in mouse models are also dysregulated in the human disease. We will monitor microcyst formation in primary cultures of human ADPKD cells following pharmacological or genetic manipulation using lentiviruses expressing shRNA to alter cilia phenotype and Hh status. Additionally, using Hh and Ca2+ modulators and cell proliferation as a read-out, we will determine whether Hh signaling interconnects with Ca2+ signaling, which is perturbed in ADPKD. Together, these experiments will reveal a novel role for Hh signaling and a potential therapeutic target for ADPKD. These experiments will also provide a convergent network of Hh signaling and known pathways in renal cystogenesis, contributing to the systems biology of renal cystic disease and shedding light on the role of primary cilia in maintaining renal tubular integrity.
The molecular process of renal cystogenesis is complex and proposed to originate from an underlying ciliary defect. This proposal aims to determine a novel role for ciliary-mediated Hedgehog (Hh) signaling in Autosomal Dominant Polycystic Kidney Disease (ADPKD) and to define the molecular mechanisms by which increased Hh signaling causes renal cysts, providing a platform for the design of novel therapeutic strategies against ADPKD.
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