BASIC RESEARCH www.jasn.org Downregulating Hedgehog Signaling Reduces Renal Cystogenic Potential of Mouse Models † † Pamela V. Tran,* George C. Talbott,* Annick Turbe-Doan,* Damon T. Jacobs, † † † Michael P. Schonfeld, Luciane M. Silva, Anindita Chatterjee, Mary Prysak,* † ‡ Bailey A. Allard, and David R. Beier* *Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; †Department of Anatomy and Cell Biology and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas; and ‡Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington ABSTRACT Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 atten- uated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target. J Am Soc Nephrol 25: 2201–2212, 2014. doi: 10.1681/ASN.2013070735 Cystic kidney disease represents a wide disease Primary cilia project from the apical surface of spectrum characterized by fluid-filled cysts, which renal tubular epithelial cells and have been proposed destroy surrounding renal parenchyma. The spec- tofunction asmechanosensors, bending in response trum affects adults in the most common life- to urine flow and initiating signaling cascades.4 In- threatening hereditary disease, autosomal dominant traflagellar transport (IFT), the bidirectional trans- polycystic kidney disease (ADPKD), as well as chil- port of multiprotein complexes along the ciliary dren in the form of autosomal recessive PKD microtubule-based core, builds and maintains cilia (reviewed by Torres and Harris1). Infantile or juvenile and is integral to signaling.5 Anterograde IFT cystic kidney disease is also manifested in disease syndromes caused by mutation of ciliary genes that are collectively termed ciliopathies. These include Received July 15, 2013. Accepted January 9, 2014. nephronophthisis, Bardet-Biedl syndrome, Meckel- Published online ahead of print. Publication date available at Gruber syndrome, Jeune syndrome, and Joubert www.jasn.org. syndrome.2 PKD genes encode proteins that also Correspondence: Dr. Pamela V. Tran, Department of Anatomy 3 fi localize to primary cilia. Together, these ndings and Cell Biology and The Kidney Institute, University of Kansas have led to the proposition that perturbed cilia func- Medical Center, 3901 Rainbow Boulevard, MS #3038, Kansas tion may be a unifying etiologic basis for cystic kid- City, KS 66160. Email: [email protected] ney disease. Copyright © 2014 by the American Society of Nephrology J Am Soc Nephrol 25: 2201–2212, 2014 ISSN : 1046-6673/2510-2201 2201 BASIC RESEARCH www.jasn.org delivers proteins to the ciliary distal tip and is mediated largely complex B genes, including a hypomorphic allele of Ift8819 by the kinesin motor and IFT complex B proteins, while ret- and renal-specificablationofIft20 or of Kif3a, which rograde IFTreturns proteins to the base and is mediated by the encodes a kinesin subunit, causes renal cysts.20–22 Because cytoplasmic dynein-2 motor and IFT complex A proteins. loss of complex B proteins results in loss of Hh response, In mice, loss of IFT proteins causes cilia structural defects, this may account in part for why Hh signaling has not been which affect regulation of Hh signaling,6 a pathway fundamental studied extensively in cystic kidney disease. to proper development and tissue maintenance.7 Three mam- One report has characterized the role of IFT complex A malian Hh ligands—Sonic Hh, Indian Hh, and Desert Hh— proteins in renal function; kidney-specific deletion of Ift140 can initiate signaling upon binding the 12-transmembrane re- caused renal cysts.23 However, THM1 mutations have been ceptor, Patched, in the cilium. Upon being bound, Patched reported in 5% of patients with ciliopathies, rendering exits the cilium, enabling ciliary translocation of the seven- THM1 the most commonly mutated gene in ciliopathies24 transmembrane signal transducer, Smoothened (SMO).8,9 and thereby implicating this gene in renal disease. We inves- Within the cilium, SMO is activated, which culminates in ac- tigated the consequences of Thm1 deletion in renal cystogen- tivation of full-length glioblastoma (GLI1, GLI2, GLI3) tran- esis using the Thm1aln/aln developmental mutant and a newly scription factors. In most tissues, GLI2 acts as the primary developed Thm1 conditional knockout mouse. We further transcriptional activator (reviewed by Eggenschwiler and examined a direct role of Hh signaling in this and two other Anderson10), although in certain tissues, GLI3 activator orthologous cystic kidney disease mouse mutants, in part by (GLI3A) or GLI3 repressor (GLI3R), which is formed by examining effects of Hh inhibitors on cystogenic potential of cleavage of the full-length GLI3 protein, plays a predominant mutant kidneys cultured with cAMP, an assay proposed as a role because of redundancy and/or tissue specificity of the GLI general model of cystic kidney disease25 and useful for testing proteins.11,12 Nonetheless, the balance between activity of GLI efficacy of potential pharmaceutical therapies.26 activators and GLI3 repressor determines level of Hh signaling output within a cell.10,13 Despite the connection between primary cilia and Hh RESULTS signaling, a role for Hh signaling in cystic kidneydisease has not been studied extensively. Yet the few studies implicating Hh Loss of THM1 Causes Renal Cysts signaling in cystic kidney disease are quite compelling. In the To explore a role for Thm1 deletion in renal cystogenesis, we first, 50% of Shh-null mouse embryos formed a single, ectopic, examined the kidneys in Thm1aln/aln mice during late embryo- dysplastic kidney.14 In the second, loss of the transcription genesis. At E16.5 and P0, the last day on which Thm1aln/aln factor, Glis2, a member of the Kruppel-like C2H2 zinc finger mutants survive, histologic analysis of the kidneys revealed protein subfamily, which includes the GLI proteins, resulted in cystic dilations of glomeruli and surrounding tubules (Figure nephronophthisis in humans and mice.15 Expression profiling 1A). Coimmunostaining of kidney sections for Lotus tetrago- 2 2 of Glis2 / kidneys revealed an upregulation of Gli1, a direct nolobus lectin (LTL) and Na+K+ ATPase (a6F) demonstrated target of the pathway, suggesting upregulated Hh signaling. that Thm1aln/aln renal tubular dilations originate in proximal Finally, a study examining the effects of corticosteroid over- tubules and ascending loops of Henle (Figure 1B). exposure on metanephric development found that adding hy- Because Thm1aln/aln mutants die shortly after birth, we gen- drocortisone to a metanephros organ culture unexpectedly erated Thm1 conditional knockout (cko) mice to analyze the caused renal cysts as well as upregulated Ihh transcripts.16 role of THM1 in maintaining renal tubular integrity. Ubiqui- Addition of Hh inhibitor, cyclopamine, reduced hydrocortisone- tous deletion of Thm1 at E17.5 resulted in cystic kidney disease induced cysts without affecting organ growth, implicating in- in 6-week-old Thm1 cko mice (Figure 1C), with elevated ratios creased Hh signaling in this mechanism of cystogenesis. of percentage kidney weight to body weight (%KW/BW) and We identified an N-ethyl-N-nitrosourea–derived, develop- BUN levels (Figure 1, D and E). Levels of cAMP were also mental mouse mutant, alien (aln),asthefirst IFT complex A higher in Thm1 cko cystic kidneys (Figure 1F), similar to other mammalian mutant.17 This defect impaired retrograde IFT, mouse models, such as the cpk autosomal recessive PKD causing accumulation of proteins in bulb-like structures at the model and the Pkd1 orthologous ADPKD model. Thm1 cko ciliary distal tips and increased Hh signaling mediated by en- renal cysts labeled positively for LTL, Tamms–Horsfall protein hanced GLI2 and GLI3 activator transcriptional activities re- (THP), and Dolichus biflorus agglutinin (DBA), indicating that sulting in limb and neural tube patterning defects. The genetic cysts originate from proximal tubules, loops of Henle, and lesion in alien results in the absence of Thm1 (TPR-containing collecting ducts, respectively (Figure 2, B–D). In kidneys of Hh modulator 1; also termed Ttc21b), an ortholog of Chlamy- Thm1 cko mice, primary cilia were stunted and showed accu-