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Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction

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Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction BASIC RESEARCH www.jasn.org Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction Sepideh Sheybani-Deloui,1,2 Lijun Chi,1 Marian V. Staite,1,2 Jason E. Cain,1 Brian J. Nieman,3,4,5,6 R. Mark Henkelman,4,5 Brandon J. Wainwright,7 S. Steven Potter,8 Darius J. Bagli,1,2,9 Armando J. Lorenzo,9 and Norman D. Rosenblum1,2,10,11 1Program in Developmental and Stem Cell Biology, 10Division of Nephrology, 3Program in Physiology and Experimental Medicine, and 9Division of Urology, The Hospital for Sick Children, Toronto, Ontario, Canada; 2Departments of Physiology, 4Medical Biophysics and Medical Imaging, and 11Paediatrics, University of Toronto, Toronto, Ontario, Canada; 5Mouse Imaging Centre, Toronto Centre for Phenogenomics Toronto, Ontario, Canada; 6Ontario Institute for Cancer Research, Toronto, Ontario, Canada; 7Genomics of Development and Disease Division, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia; and 8Department of Pediatrics, Cincinnati Children’s Hospital, Cincinnati, Ohio ABSTRACT Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI- dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine in- termediate mesoderm–derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. + Whole transcriptome analysis of isolated Ptch2 cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm–derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that ob- served in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction. J Am Soc Nephrol 29: 532–544, 2018. doi: https://doi.org/10.1681/ASN.2017050482 Ureteropelvic junction obstruction (UPJO) is the Received May 4, 2017. Accepted October 5, 2017. most common form of congenital urinary tract ob- S.S.-D. and L.C. contributed equally to this work. struction. UPJO is also a major cause of antenatal fi Published online ahead of print. Publication date available at dilation of the renal pelvis, identi ed by ultrasound www.jasn.org. in 1% of fetuses.1 Most cases of UPJO are caused by Correspondence: Dr. Norman D. Rosenblum, The Hospital for intrinsic narrowing of the ureter at the pelvic-kidney Sick Children, Peter Gilgan Centre for Research and Learning, Rm junction, with deposition of extracellular matrix, in- 16-9-706, 686 Bay Street, Toronto, ON M5G 0A4, Canada. Email: filtration of inflammatory cells, and smooth muscle [email protected] – hypertrophy.2 4 Yet, underlying pathogenic Copyright © 2018 by the American Society of Nephrology 532 ISSN : 1046-6673/2902-532 J Am Soc Nephrol 29: 532–544, 2018 www.jasn.org BASIC RESEARCH mechanisms are largely undefined, particularly because of the Statement of Significance absence of experimental models of congenital UPJO. Lack of reli- able biomarkers to differentiate UPJO from other forms of kidney Ureteropelvic junction obstruction (UPJO) is a major cause of con- dilation limits the ability to predict a need for corrective surgery, genital dilation of the renal pelvis. The pathogenesis of UPJO is fi fi currently performed in approximately 50% of affected infants,5,6 unde ned, no biomarkers are identi ed to predict its natural history, and surgical correction is the only treatment. This study demon- fi 2–4 and the development of speci c therapeutic strategies. strates that increased Hedgehog signaling activity, caused by Ptch1 The ureteropelvic junction (UPJ) is a component of the deficiency within a narrow time window during mouse develop- renal collecting system, which consists of collecting ducts, ca- ment, results in ectopic localization of cortical stromal cells to the lyces, and the pelvis, all derived from the epithelial ureteric bud ureteropelvic junction, causing blockage of urinary outflow and (UB). After UB outgrowth from the Wolffian duct at 5 weeks dilation of the renal pelvis. Expression of genes characteristic of cortical stromal cells and Hedgehog signaling activity was increased human gestation (approximately E10.5 in mice), the distal UB in obstructive ureteric tissue resected from affected children. These elongates caudally and integrates with the bladder.7,8 The ros- results provide a basis for genetic studies in human UPJO and tral tip of the UB invades the metanephric mesenchyme (MM) therapeutic strategies targeting the Hedgehog signaling pathway. and undergoes repeated branching events regulated by recip- rocal inductive interactions between nephron progenitors sur- during murine renal development.26,28 Embryonic lethality rounding the UB tips, and Foxd1+ cells that surround the before metanephric kidney development in Ptch1-null mice nephron progenitors.9–12 Renal calyces and the pelvis are provides a basis to investigate Ptch1 functions in a lineage-specific formed from the first several ureteric branch generations.13 manner.29 We generated mice in which Ptch1 deficiency is re- Although the morphogenetic events in pelvicalyceal remodel- fl fl stricted to the metanephric lineage using Ptc1 ox/ ox mice and an ing are largely undefined, Wnt7b expression in the ureteric 2 2 2 2 MM-specific Rarb2Cre allele (Ptch1 / MM).30–32 Ptch1 / MM stalk is crucial in ureteric tree elongation and pelvis forma- tion.14 Ureteric smooth muscle development begins at E15.5 mice showed a 60% decrease in expression of Ptch1 exon 3, the with differentiation of Tsh3+ and Tbx18+ smooth muscle pro- target of CRE-mediated excision, in metanephric rudiments at , genitors, both of which arise from tail-bud mesoderm.15–17 E11.5 (n=3; P 0.05; Figure 1A). qRT-PCR analysis of an undis- During renal development, the Hedgehog (HH) ligand, Sonic rupted exon of Ptch1 (exon 17) demonstrated a two-fold increase , hedgehog (SHH), and its receptor Patched 1 (Ptch1), are expressed as early as E11.5 (n=3; P 0.05; Figure 1B), consistent with in tubular epithelium and stromal cells adjacent to the pelvic- increased HH signaling activity. Compared with control mice, kidney junction. In vertebrates, HH ligand binds to PTCH and expression of the Ptc1-lacZ reporter allele was increased in the 2/2MM relieves PTCH-mediated inhibition of Smoothened, a transmem- medullary stroma and expanded to the renal cortex in Ptch1 brane protein expressed on the cell surface.18,19 In the presence of kidneys (Figure 1, C and D). 2/2MM HH, full-length GLI proteins translocate to the nucleus, acting as Histologic analysis of Ptch1 kidneys at E18.5 dem- transcriptional activators.20,21 In the absence of HH, full-length onstrated distention of the renal pelvis (i.e., hydronephrosis) GLI3 is processed to a truncated N-terminal form (GLI3R), which bilaterally and ablation of the renal medulla with no ureteric acts as a transcriptional repressor.22,23 In mice, GLI3R regulates dilation (80% penetrance, 29 males and 33 females) (Figure 1, ureteric branching, nephron progenitor proliferation, and urinary E and F). Measurement of pelvic volume in three-dimensional pacemaker activity.24–27 SHH acts as a paracrine signal to Tbx18+ images of E18.5 mutant kidneys generated by magnetic reso- mesenchymal cells to control smooth muscle differentiation and nance imaging (MRI) revealed a nine-fold increase (n=10; proliferation.16,28 P,0.05; Figure 1, G–I). Histologic and immunofluorescence Here, we identified increased HH-GLI signaling as a cause of analysis of the nascent epithelial collecting ducts and distal congenital UPJO in mice and a marker of UPJO in pediatric tubules marked, respectively, by calbindin and E-cadherin, patients. Ptch1 deficiency in the MM caused congenital intrinsic demonstrated comparable formation of the epithelial tubular UPJO because of an ectopic population of PTCH2+ stromal pro- network within the renal medulla in both wild-type (WT) and genitor cells arising from intermediate mesoderm (IM)–derived mutant kidneys at E15.5 (Supplemental Figure 1, A–F). Fur- kidney progenitors at the onset of MM specification. Remarkably, ther, expression of LEF1, a canonical WNT pathway effector expression of stromal and HH signaling-related genes was in- that is expressed in medullary interstitial cells and regulates creased in obstructing ureter tissue segments
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