BASIC RESEARCH www.jasn.org Integrin-linked Kinase Controls Renal Branching Morphogenesis via Dual Specificity Phosphatase 8 †‡ Joanna Smeeton,* Priya Dhir,* Di Hu,* Meghan M. Feeney,* Lin Chen,* and † Norman D. Rosenblum* § *Program in Developmental and Stem Cell Biology, and §Division of Nephrology, The Hospital for Sick Children, Toronto, Ontario, Canada; and †Departments of Paediatrics, and ‡Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada ABSTRACT Integrin-linked kinase (ILK) is an intracellular scaffold protein with critical cell-specific functions in the embryonic and mature mammalian kidney. Previously, we demonstrated a requirement for Ilk during ureteric branching and cell cycle regulation in collecting duct cells in vivo. Although in vitro data indicate that ILK controls p38 mitogen-activated protein kinase (p38MAPK) activity, the contribution of ILK- p38MAPK signaling to branching morphogenesis in vivo is not defined. Here, we identified genes that are regulated by Ilk in ureteric cells using a whole-genome expression analysis of whole-kidney mRNA in mice with Ilk deficiency in the ureteric cell lineage. Six genes with expression in ureteric tip cells, including Wnt11, were downregulated, whereas the expression of dual-specificity phosphatase 8 (DUSP8) was upregulated. Phosphorylation of p38MAPK was decreased in kidney tissue with Ilk deficiency, but no significant decrease in the phosphorylation of other intracellular effectors previously shown to control renal morphogenesis was observed. Pharmacologic inhibition of p38MAPK activity in murine inner med- ullary collecting duct 3 (mIMCD3) cells decreased expression of Wnt11, Krt23,andSlo4c1.DUSP8over- expression in mIMCD3 cells significantly inhibited p38MAPK activation and the expression of Wnt11 and Slo4c1. Adenovirus-mediated overexpression of DUSP8 in cultured embryonic murine kidneys decreased ureteric branching and p38MAPK activation. Together, these data demonstrate that Ilk controls branching morphogenesis by regulating the expression of DUSP8, which inhibits p38MAPK activity and decreases branching morphogenesis. J Am Soc Nephrol 27: 1465–1477, 2016. doi: 10.1681/ASN.2015020139 Renal branching morphogenesis, defined as growth contrast, a limited number and variety of intracellular and branching of the ureteric bud (UB) and its molecules that function within these signaling path- derivatives, is essential to mammalian kidney devel- ways to control ureteric branching have been identi- opment. The UB grows, branches, and differentiates fied. In vitro treatment of embryonic urogenital to form the collecting ducts and pelvis of the mature explants with pharmacologic inhibitors revealed dis- kidney. Further, UB tips induce adjacent metanephric tinct roles for extracellular signal-regulated kinase mesenchyme cells to undergo the process of nephro- genesis.1 Defects in renal branching morphogenesis cause congenital renal hypodysplasia, characterized Received February 6, 2015. Accepted August 11, 2015. by abnormal collecting-system morphology and Published online ahead of print. Publication date available at 2 function and low nephron number. www.jasn.org. TheUBarisesasadirectevaginationofthein- fi Correspondence: Dr. Norman D. Rosenblum, The Hospital for termediate mesoderm-derived Wolf an duct. Ex- Sick Children, Peter Gilgan Centre for Research and Learning, tracellular ligands and cell-surface receptors in mul- 686 Bay Street, Toronto, ON M5G 0A4, Canada. Email: norman. tiple distinct molecular signaling pathways function [email protected] during the induction and early patterning of the UB. In Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 27: 1465–1477, 2016 ISSN : 1046-6673/2705-1465 1465 BASIC RESEARCH www.jasn.org (ERK), protein kinase B (AKT), and p38 mitogen-activated pro- tein kinase (p38MAPK) during renal development.3 Further- more, ERK and AKT can be activated downstream of the tyrosine kinase receptor, RET, in the UB tip domain, suggesting that in- tracellular kinase signaling is functionally important during ure- teric branching.4,5 Integrin-linked kinase (ILK) is a scaffold protein that was initially identified through its ability to interact with the cytoplasmic domain of b-integrins.6 Investigation of ILK function has revealed a requirement for ILK upstream of in- tracellular kinases AKT,7 glycogen synthase kinase 3b,8 and p38MAPK.9 While each of AKT, glycogen synthase kinase 3b, and p38MAPK have been shown to play a role in control- ling renal branching morphogenesis, their role downstream of ILK in the embryonic kidney has not been defined. Nor is it predicted by their functions in nonrenal tissues because their regulation by ILK is context-dependent.10 Previously, we dem- onstrated that Ilk is required for renal branching morphogen- esis in vivo11 and controls UB branching via p38MAPK in vitro.9,12 However, the role of p38MAPK in vivo and the genes that act downstream of Ilk during the early stages of branching morphogenesis have not been previously defined. In this study we characterize the intracellular signaling pathways and UB transcriptome in the early embryonic Ilk- deficient kidney to determine the primary role of Ilk in the regulation of renal branching. Our results demonstrate that Ilk regulates the expression of a distinct subset of UB branching 2 2 genes through both p38MAPK-independent and -dependent Figure 1. E12.5 Ilk / UB kidneys demonstrate reduced UB pathways. Our data also demonstrate upregulation of a phos- branching and abnormal gene regulation. (A,B) GFP branching 2/2UB phatase, dual-specificity phosphatase 8 (DUSP8), not previ- pattern of (A) GFP+ heterozygote and (B) Ilk kidneys con- fi 2/2UB ously implicated in kidney development. We demonstrate a rmed the branching defect in E12.5 Ilk kidneys selected for microarray analysis. (C) Gene expression differs between WT functional role for DUSP8 in attenuating activation of 2/2UB and Ilk mutant (MUT) kidneys with similar expression pat- p38MAPK and p38MAPK-dependent genes and inhibiting terns clustering within the WT and mutant samples. Expression branching morphogenesis. Together, these results describe a values of differentially regulated genes (P,0.003) are shown in novel DUSP8-mediated mechanism by which ILK controls this heatmap, with red indicating lower expression and yellow renal branching morphogenesis. higher expression. RESULTS To determine changes in expression levels for individual genes, the top table of genes based on the t-statistic ranking was gen- Mice with conditional deletion of Ilk in the developing UB erated. Using a statistical cutoff of P,0.003 to identify changes 2 2 (Ilk / UB) have decreased UB branching at E12.5.11 However, in the expression of individual genes, we identified 131 down- the underlying molecular mechanisms are unknown. We in- regulated probe sets and 96 upregulated (Figure 1C, Supple- vestigated these mechanisms using whole-genome–based anal- mental Tables 2 and 3). The magnitude of changes in gene ysis of gene expression in intact kidney tissue isolated at E12.5 expression, particularly for downregulated genes, was twofold (n=6 kidneys per sample, three biologic replicates per geno- or lower. This may be due to the fact that UB gene expression type). At the time of dissection, the presence of a branching was assayed in whole tissue in which UB cells constitute a mi- phenotype in Ilk-deficient kidneys was documented using nority of cells. While a gene expression analysis in isolated UB green fluorescent protein (GFP) expression driven by the cells may have generated gene expression changes of higher Hoxb7 promoter (Figure 1). Gene expression changes were in- magnitude, the quality of RNA isolated from flow-sorted UB vestigated through microarray analysis using Affymetrix cells derived from mutant mice precluded a whole-genome GeneChip Mouse 430 2.0 arrays. Expression levels were nor- gene expression analysis. Notwithstanding these considera- malized to wild-type (WT) and then analyzed for differential tions, consistent with genetic deletion of Ilk, the most statisti- 2 2 expression between Ilk / UB and WT samples using cally significant gene expression change was a downregulation 2 2 the Bioconductor limma package in R statistical program.13 of Ilk in Ilk / UB kidneys. 1466 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1465–1477, 2016 www.jasn.org BASIC RESEARCH Gene Ontology Analysis Suggests Abnormal statistical significance for six genes by qRT-PCR (n=6/genotype, Regulation of Tube Morphogenesis Genes two technical replicates) and these were further confirmed by in To define the global state of gene expression in an Ilk-deficient situ hybridization in tissue sections at E12.5 (Figure 2). In con- state, we performed pathway analysis to identify gene ontology trast to UB tip genes Ret and Sox9, the mRNA expression of which (GO) biologic processes that are enriched within the down- was not significantly altered, in situ hybridization analysis con- regulated gene set. Sixty-one biologic process GO terms were firmed decreased expression of Sox8, Wnt11, Myb, CXCR4, Krt23, 2 2 statistically enriched in the downregulated gene list (Supple- and Slco4c1 in the UB of E12.5 Ilk / UB kidneys (Figure 2). mental Table 4). Within the downregulated gene set, GO terms involved in epithelial tube development as well as the regula- Ilk Deficiency Causes a Specific Decrease in the tion of organ morphogenesis were enriched (Table 1). These Activation of p38MAPK terms are highly consistent