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Novel Insights into the Pathogenesis of Monogenic Congenital Anomalies of the and Urinary Tract

Amelie T. van der Ven, Asaf Vivante, and Friedhelm Hildebrandt

Divison of Nephrology, Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts

ABSTRACT Congenital anomalies of the kidneys and urinary tract (CAKUT) comprise a large an entirely unblemished contralateral spectrum of congenital malformations ranging from severe manifestations, such urinary system.10 as renal agenesis, to potentially milder conditions, such as vesicoureteral reflux. Thediverse manifestationsof CAKUT CAKUT causes approximately 40% of ESRD that manifests within the first three phenotypes are thought to result from decades of life. Several lines of evidence indicate that CAKUT is often caused by disturbancesatany pointinrenalmorpho- recessive or dominant mutations in single (monogenic) . To date, approxi- genesis (Figure 1).15,16 Most importantly, mately 40 monogenic genes are known to cause CAKUT if mutated, explaining imbalances in the communication be- 5%–20% of patients. However, hundreds of different monogenic CAKUT genes tween the metanephric mesenchyme probably exist. The discovery of novel CAKUT-causing genes remains challenging (MM) and the ureteric bud (UB) are because of this pronounced heterogeneity, variable expressivity, and incomplete believed to be central to the pathogene- penetrance. We here give an overview of known genetic causes for human CAKUT sis of CAKUT phenotypes (Figure 1, and shed light on distinct renal morphogenetic pathways that were identified as C–E).15 The developmental origin of relevant for CAKUT in mice and humans. PUVs, however, likely differs from other CAKUT manifestations and remains J Am Soc Nephrol 29: 36–50, 2018. doi: https://doi.org/10.1681/ASN.2017050561 poorly understood.3,17,18 We have previously hypothesized that a high fraction of human CAKUT may be caused by single- defects. Congenital anomalies of the kidneys upper urinary tract (e.g., renal agenesis Monogenic diseases, also known as Men- and urinary tract (CAKUT) are com- and renal hypodysplasia) to phenotypes delian disorders, are caused by mutations mon malformations with a potentially primarily affecting the lower urinary in a single causative gene.10 Supporting fl severe effect on health. CAKUT causes tract, such as vesicoureteral re ux evidence for a monogenic etiology in about 40% of cases of ESRD in patients (VUR), ureterovesical junction obstruc- the case of CAKUT comes from (1)the fi who develop it within the rst three tion (UVJO), and posterior urethral familial occurrence of CAKUT pheno- 1,2 3,8,11 decades of life. Manifestations of valves (PUVs). In addition, abnor- types (approximately 10%–15% of pa- the CAKUT spectrum account for malities of kidney shape or anatomic tients with CAKUT cases are familial), – 20% 30% of congenital malforma- position, such as horseshoe kidney or (2) the existence of monogenic syn- 3–7 tions and constitute a frequent cause pelvic kidney, may occur. Malformations, dromes with a distinct syndromic phe- of birth defects (approximately three such as UVJO, PUV, and VUR, may lead notype that includes manifestations 4,8,9 to six per 1000 live births). CAKUT to anatomic (UVJO and PUV) or func- from within the CAKUT spectrum may occur either as an isolated condi- tional (VUR) stasis of urine flow, tion or along with extrarenal manifesta- thereby increasing the risk of urinary tions as part of a syndromic disorder.10–13 tract infections, which may result in re- Published online ahead of print. Publication date To date, .200 clinical syndromes have nal scarring and potentially, CKD (e.g., available at www.jasn.org. been described that comprise fea- from reflux nephropathy). It is impor- Correspondence: Prof. Friedhelm Hildebrandt, tures of CAKUT as part of their distinct tant to note that, although different Boston Children’s Hospital, Division of Nephrol- ogy, EN561, 300 Longwood Avenue, Boston, MA 14 phenotype. manifestations from within the CAKUT 02251. Email: Friedhelm.Hildebrandt@childrens. The term CAKUTsummarizes a large spectrum may coexist in the same indi- harvard.edu 3,15 variety of diverse congenital malforma- vidual, unilateral CAKUT (e.g., renal Copyright © 2018 by the American Society of tions that range from conditions of the agenesis) can also exist in the presence of Nephrology

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(.200 to date), (3)theexistenceof monogenic mouse models that exhibit CAKUT (.180 to date), and (4)the fact that the development of the kidney and urinary tract is governed by distinct developmental genes.10,11,19,20 Approximately 40 different monogenic causes for human CAKUT (25 dominant and 15 recessive) have so far been identi- fied (Table 1).12,13,21–51 Many established CAKUT genes encode transcription fac- tors and follow a dominant pattern of in- heritance.3,10 Some of the genes listed in Table 1, however, remain questionable in regard to their pathogenic effect and will require more support through functional studies in the future.52,53 Currently, at most, up to 18% of CAKUT cases can be explained by these established monogenic causes (Table 1).3,10 The percentage rate of molecu- larly “solved” cases, of note, is hereby likely dependent on the corresponding selection of patients and controls. Ap- proximately 10%–15% of cases have ad- ditionally been described to occur due to copy number variations that predomi- nantly involve either the HNFB1 locus on 17 or the Di-George/ velocardiofacial syndrome region on chromosome 22.54 It is likely that hundreds of additional Figure 1. Development of the kidneys and urinary tract. (A) The bilateral nephric ducts (NDs; monogenic causes of human CAKUT alternatively mesonephric ducts or Wolffian ducts) and the nephric cords (NCs) are the precursor have yet to be identified. Apart from structures of the adult urinary system. Both originate from the embryonic intermediate mesoderm. The cells of the ND undergo an early mesenchymal to epithelial transition and assemble into the distinct heterogeneity, with 40 genes fi fi epithelial tube–like structures. The associated NC retains characteristics of mesenchymal tis- identi ed so far (Table 1), the identi - sue.16,62 (B) As the embryo develops, the ND elongates caudally. At approximately E9.5, the most cation of novel CAKUT-causing genes caudal portion of the ND fuses with the cloacal epithelium (Cl). The cloaca is the embryonic is complicated by variable expressivity precursor of the bladder, and it is derived from cloacal endoderm.16,66 (C) The NC reorganizes and incomplete penetrance.10,11 These and forms a morphologically distinct domain: the MM. Renal morphogenesis is initiated and features are frequently encountered in maintained by reciprocal interactions between the epithelial ND and the MM. In mice, at em- dominant diseases and can result in bryonic day approximately E10–E10.5, signals from the MM induce the formation of a circum- the phenomenon that either individu- 15,16 scribed, broad swelling of the ND at the level of the MM. (D) At E10.5 in mice and around the als carrying a mutation in a CAKUT fi fth week of human gestation, the UB emerges from the swollen portion of the ND and grows gene can present with a phenotype dorsally toward the MM.4,15,16,74 The caudal part of the ND, which is located between the UB and that differs from the phenotypic mani- the insertion into the Cl, is referred to as the common nephric duct (CND). (E) Stimulated by MM- derived signals, the UB begins to branch repeatedly (branching morphogenesis) at approximately festation of other individuals with an E11.5. Through continuous reciprocal induction, the MM is important for promoting and main- identical mutation (variable expressiv- taining branching events of the UB. The UB branching continues for approximately 9–13 cycles ity) or alternatively, an individual (mice) and then slows down after approximately E15.5. Via branching morphogenesis, the UB carrying a mutation does not exhibit a gives rise to the renal collecting system consisting of collecting ducts and renal pelvis as well as the CAKUT phenotype at all (incomplete ureter. Reciprocally, signals from the UB also support development of MM cells. The MM that is in penetrance).10 closest proximity to the UB tips condenses and forms the so-called cap mesenchyme (CM). One theory proposed by Ichikawa Stimulated by signals from the UB, the CM undergoes a mesenchymal to epithelial transition. The et al.15 suggests that this phenotypic epithelial cell population subsequently gives rise to structures of the nephron (glomerulus, the variability may occur as a result of (1) fi proximal tubule, and the distal tubule). Modi ed from refs. 16 and 62, with permission. stochastic spatiotemporal differences

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Table 1. Fifteen recessive and 25 dominant genes that represent monogenic causes of human CAKUT if mutated Gene Refs. Autosomal recessive ACE I–converting enzyme 38 AGT Angiotensinogen 38 AGTR1 Angiotensin II , type 1 38 CHRM3 Muscarinic acetylcholine receptor M3 39 FRAS1 ECM protein FRAS1 42 FREM1 FRAS1-related ECM protein 1 42 FREM2 FRAS1-related ECM protein 2 42 GRIP1 Glutamate receptor interacting protein 1 42 HPSE2 Heparanase 2 (inactive) 43 ITGA8 Integrin-a8 44 LRIG2 -rich repeats and Ig-like domains 2 45 REN 38 TRAP1 Heat shock protein 75 (also known as TNF receptor–associated protein 1) 46 Autosomal dominant BMP4 Bone morphogenic protein 4 21 CHD1L Chromodomain helicase DNA binding protein 1 like 22 CRKL CRK-like proto-oncogene, adaptor protein 23 DSTYK Dual serine/threonine and tyrosine protein kinase 24 EYA1 absent homolog 1 25 GATA3 GATA binding protein 3 26,188 HNF1B HNF B 13 MUC1 Mucin 1 27 NRIP1 interacting protein 1 140 PAX2 Paired box 2 12 PBX1 PBX homeobox 1 53 RET Proto-oncogene tyrosine-protein kinase receptor Ret 28 ROBO2 Roundabout, axon guidance receptor, homolog 2 (Drosophila) 29,189 SALL1 Sal-like protein 1 (also known as spalt-like 1) 49 SIX2 SIX homeobox 2 21 SIX5 SIX homeobox 5 51 SLIT2 homolog 2 29 SOX17 Transcription factor SIX-17 30 SRGAP1 SLIT-ROBO Rho GTPase activating protein 1 29 TBX18 T-box transcription factor TBX18 31 TNXB Tenascin XB 32 UMOD Uromodulin 33 UPK3A Uroplakin 3A 34 WNT4 Protein Wnt-4 35–37 X-linked recessive KAL1 Anosmin 1 47 during key events of renal morphogene- factors potentially provide a sufficient COMPLEX GENETICS IN CAKUT sis, (2)genedosageeffects,and(3) explanation for interindividual differ- redundancy of genes that belong to ences in CAKUT manifestations, they are Nowadays, modern study designs increas- functionally related gene families.10,20 less applicable in the context of intraindi- ingly yield potential polygenic disease loci This hypothesis thereby provides an ex- vidual (left-right) dissimilarities. Partic- for a wide variety of human disease condi- planation for both intraindividual as ularly, environmental factors are tions.61 Despite the complicated etiology well as interindividual (i.e.,variableex- extremely interesting to discover, because of CAKUT, which suggests an interplay of pressivity) differences in CAKUT phe- they may allow for a specific prophylaxis (epi-)genetic and environmental fac- notypes. Alternative hypotheses ascribe of CAKUT. However, despite large stud- tors,3,62 to date, there is little evidence for more importance to epigenetic or envi- ies, to date, only little success has been the simultaneous contribution of more ronmental influences (e.g.,effectsof achieved regarding the search for environ- than one gene (di-, oligo-, or polygenic malnutrition, vitamins, or drugs).3,55 mental factors with significant effect on effects; i.e., complex genetics) to the path- Although epigenetic and environmental the pathogenesis of CAKUT.3,56–60 ogenesis of human CAKUT.63 Similar to

38 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 36–50, 2018 www.jasn.org BRIEF REVIEW monogenic research, the identification of or two components of the paralogous RET signaling pathway are beyond the polygenic causes of CAKUT is likely com- group (i.e., Hoxa11, Hoxd11,and scope of this review and have been re- plicated by the vast heterogeneity of Hoxc11; no discernible kidney abnormal- viewed in detail recently.4,16,62,65,74,75 CAKUT. This results in very few affected ities or renal hypoplasia, respectively).64 However, variousadditional morpho- patients with shared disease loci essential The insights from mouse models, genetic pathways have previously for the unequivocal identification of novel however, do not always directly trans- been implicated to be essential for proper genetic regions. General awareness of the late to human genetics. Explanations renal morphogenesis. These signaling possibility of complex inheritance patterns for this discrepancy include potential pathways include the BMP (TGFb), in CAKUT is necessary in the genetic eval- species-specific differences in the devel- NOTCH, Hedgehog-GLI, and FGF-related uation of affected individuals. Polygenic opment of the kidneys and urinary cascades as well as canonical WNT/ disease hypotheses, however, remain chal- tract, alterations in the required gene b-catenin signaling that converge and lenging to establish, test, and prove in hu- dosage during developmental steps, partially modulate one another during man CAKUT. functional compensation by redundant the development of the kidneys and genes, incomplete penetrance, and the urinary tract.76–78 likely, merely the rarity of human cases In this review, we will place special RELEVANCE OF MOUSE with mutations in a particular gene (due emphasis on two pathways that appear CANDIDATE GENES to distinct heterogeneity). interweaved with GDNF-RET signaling and are not commonly discussed in the One of the reasons why it was hypothe- context of CAKUT. These are (1)therole sized that a large fraction of human MOLECULAR PATHWAYS OF of extracellular matrix (ECM) CAKUT may be of monogenic origin RENAL MORPHOGENESIS (Supplemental Table 3) at the epithelial- was the presence of a large number of mesenchymal interface between UB monogenic mouse models of CAKUT; Despite challenges, mouse models of and the MM (Figure 2), and (2)vitamin .180 monogenic causes of murine CAKUT have rendered it possible to A/retinoic acid (RA) signaling (Figure 3). CAKUT have been described to date identify many developmental pathways This latter pathway also constitutes the (Supplemental Table 1). These genes as relevant for renal morphogenesis first molecular pathway potentially ame- constitute promising candidates in the and pathophysiologic events that under- nable to treatment. We furthermore search for (novel) genetic causes of hu- lie CAKUT.3,10,16,48 Most prominently, briefly describe (3) the BMP signaling cas- manCAKUT.Infact,manyofthe40 the glial cell–derived neurotrophic cade as an example for a well established established monogenic causes of human factor (Gdnf)-glial cell–derived neuro- molecular pathway in renal morphogene- CAKUT (e.g., FRAS1, FREM1, FREM2, trophic factor family receptor a1 sis (Figure 4, Supplemental Table 4). The GRIP1, bone morphogenic protein 4 (Gfra1)-Ret pathway plays a decisive BMP pathway represents an additional ex- [BMP4], SIX2, Ret Proto-Oncogene role in renal morphogenesis in general ample of a signaling cascade, the genes and [RET], SALL1,andUPK3A)wereini- and specifically, the intricate regulation proteins of which have recently been iden- tially derived as candidate genes from of the crosstalk between the UB and the tified as genetic causes of human and/or observations in mouse models of MM (Figure 1, C–E).4,15,62,65 RET is a murine CAKUT.21,42,79–95 CAKUT and subsequently screened for receptor tyrosine-kinase that is involved their prevalence in human disease in a multitude of developmental cohorts.21,28,34,42,49 Controlled condi- processes.65,66 In the developing kidney, CAKUT AND ECM PROTEINS tions in the evaluation of genetic mouse RET is predominantly expressed in models allow for a careful assessment of nephric duct–derived structures (in- The ECM is a highly complex, three- subtle manifestations of underlying cluding UB) (Figure 1).65,67–69 During dimensional network of proteins and genotypes (e.g.,mildphenotypesinsin- renal morphogenesis, RET requires the proteoglycans.96,97 It is an essential com- gle heterozygous carriers of recessive ligand GDNF as well as the coreceptor ponent of all tissues and synthesized by CAKUT genes). Mouse models further- GFRA1 for its activation.65,66,70–73 embryonic cells starting at the earliest more provide the opportunity for a con- Given the important role of RET sig- stages of development.96,97 Over the trolled evaluation of the contributions of naling for renal morphogenesis, it is not past decades, the understanding of the multiple genes (i.e.,complexinheri- surprising that many of the established functional roles of the ECM has changed tance) to the development of CAKUT. causes of murine and human CAKUT dramatically. Although the ECM was Oneexampleistheeffectoftheloss- (including PAX2, GATA3, EYA1,and initially believed to merely provide struc- of-function of the entire Hox11 paralo- SALL1) have been reported to function tural support,97–99 the current under- gous group on renal morphogenesis in upstream and downstream of the standing goes far beyond simple adhesion mice (complete loss of metanephric kid- GDNF-GFRA-RET regulatory pathway and space-filling properties and includes ney induction) in comparison with an (summarized in Supplemental Table regulatory roles in cell-cell and cell matrix isolated loss-of-function of individual 2).4,16,62,65,74 Mechanistic details on the interactions.96

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Figure 2. ECM proteins cause murine and human CAKUT. Schematic of the interface between the UB and the MM in renal development. Proteins encoded by genes that, if mutated, cause monogenic CAKUT in humans are highlighted in yellow. Red frames indicate proteins encoded by genes mutated in CAKUT in mice. (Left panel) FRAS1 and FREM2 localize in epithelial cells of the UB as transmembrane proteins with intracytoplasmic tail regions.41,98,103 The interaction with PDZ domains of the intracellular protein GRIP1 is essential for targeting to the basal surface of the UB cells and shedding of FRAS1 from the membrane.98,107 FREM1 is produced by the MM and secreted into the extracellular space.106,107 FRAS1, FREM2, and FREM1 assemble at the epithelial-mesenchymal interface to form the FC.107 Mutations in the genes encoding these proteins cause CAKUT (Supplemental Figure 2). Also, mutations in GRIP1 result in human and murine CAKUT. Nephronectin (NPNT) functions as an adaptor to interconnect the ternary FC with the ITGA8/integrin-b1 heterodimer on the surface of MM cells. ITGA8/integrin-b1 signaling leads to an increased expression of GDNF by the MM, thereby promoting renal morphogenesis.183 Mutations in ITGA8 in humans and mice cause CAKUT.44 Loss of FC integrity results in a significant decrease in GDNF expression in the MM, thereby hampering the interaction between the UB and the MM and consequentially, impeding renal morphogenesis.183 (Right panel) Laminins are cross-shaped ECM molecules that consist of three distinct chains (a, b,andg).101 Laminins simultaneously interact with cell surface receptors (integrins), HSPG, collagen, and nidogen 1 (see box).184 Laminins that contain g1-chains form high-affinity

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first came to attention when mutations in genes encoding members of the Fraser complex (FC) as well as the associated protein integrin-a8 (ITGA8) were iden- tified in a significant proportion of patients with isolated human CAKUT phenotypes.42,44 Figure 2 depicts se- lected ECM components that have pre- viously been shown to be causative of human (highlighted in yellow in Figure 2) as well as murine CAKUT (outlined in red in Figure 2).41–44,93,100–105 The FC is a ternary protein complex consisting of FRAS1, FREM2, and FREM1 (Figure 2).106 Although not par- ticipating in the complex formation itself, GRIP1 is a cytosolic protein essen- tial for trafficking and proper targeting of the transmembrane protein FRAS1 to the basolateral surface of the UB cells.104 After they are assembled at the epithelial-mesenchymal interface, the Figure 3. RA signaling plays a central role for the development of the kidneys and urinary members of the FC interact with nephro- tract. Genes encoding the proteins highlighted by red frames have been implicated in nectin (NPNT), which serves as an adap- murine cases of syndromic CAKUT (Supplemental Table 1). Genes highlighted in yellow, if tor for ITGA8 and integrin-b1that mutated, constitute causes of human CAKUT. Mutations in genes encoding multiple are expressed by the cells of the MM (Fig- proteins involved in intracellular RA processing and signaling have previously been iden- 105,107,108 tified to cause syndromic CAKUT in mice (Retinol Dehydrogenase 10 [Rdh10], Aldehyde ure 2). ITGA8 is an important Dehydrogenase 1 Family Member A2 [Aldh1a2], and Cytochrome P450 Family 26 [Cyp26], upstream activator of GDNF, which, via Rxr, Rara, and Rarb).138,150–152 RDH10150 catalyzes the reversible reaction from retinol to interaction with RET, promotes renal retinal. Retinal is then converted into the active metabolite all-trans RA via an irreversible organogenesis.105,107 reaction catalyzed by the enzyme ALD1A2.138 RA then either enters the nucleus or is Genes encoding members of the FC metabolized via enzymes of the CYP26 family (e.g.,CYP26A1)151,152 in the endoplasmic have previously only been known to be reticulum. In the nucleus, RA derivatives can serve as a ligand for two receptors: RXR (9-cis- causative of the severe syndromic pheno- 123,142 RA and rexinoids) and RAR (all-trans-RA). Both receptor proteins have at least three type , a rare syndromic – subtypes and isoforms each. RXR and RAR heterodimers bind to retinoic acid responsive condition with phenotypic features in- elements (RAREs) of the nuclear DNA.121,141 RAREs are predominantly located in promoter cluding CAKUTas well as cryptophthal- regions of target genes. The presence of RA recruits coactivators and leads to enhanced binding to RAREs and expression of target genes.121,141 The transcriptional control of mos and cutaneous (FRAS1, 41,103,104,109,110 downstream genes is further modified by the binding of additional coregulators (co- FREM1,andGRIP1). activators and corepressors, including NRIP1) to RXR and RAR.121,141,153 We recently FREM1 mutations are also an established identified mutations in the corepressor NRIP1 (highlighted in yellow) as causing CAKUT in genetic cause for the phenotypically humans.140 NRIP1 may interact with both RXRs and RARs.153 related conditions Manitoba-oculo- tricho-anal syndrome (OMIM 248450) Thepathophysiologicinter-relation phenotypic features of the CAKUTspec- and Bifid nose with or without anorec- of ECM components and renal morpho- trum (summarized in Supplemental Ta- tal and renal anomalies syndrome genesis has long been known from ble 3). The relevance of ECM proteins (OMIM 608990) (Supplemental Fig- knockout animal models that display for isolated human CAKUT, however, ures 1 and 2).111 interactions with nidogen 1.101 Fourteen distinct laminin isoforms have been identified to date, of which ten possess the laminin-g1 chain.101,185 A homozygous ablation of the nidogen binding site in laminin-g1 has been shown to result in severe CAKUT phenotypes (including bilateral renal agenesis) in mice.101 Despite its important role in ECM assembly, targeted inactivation of nidogen 1 did not result in a CAKUT phenotype.101 Both nidogen 1 and Laminin are known interactors of HSPG. HPSE is one of the few enzymes with the ability to break down HSPG.115 Knockdown of neither HSPG nor HPSE has been reported to cause CAKUT phenotypes in mice.115,120,186,187 However, recessive mutations in HPSE2 have been identified in human patients with urofacial syndrome,43,93 with a similar phenotype in mice.43 Although HPSE2 has no detectable enzymatic activity itself, it has been shown to function as an endogenous inhibitor of HPSE (Supplemental Figure 3).116 Modified from refs. 107 and 184, with permission.

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CAKUT phenotypes (Figure 2).42 These findings thereby indicate, that, in the case of the FC genes, the disease manifes- tation can vary from severe syndromic phenotypes on the basis of truncating mu- tations to isolated CAKUTon the basis of hypomorphic mutants (e.g., missense) (Supplemental Figure 2). Another subgroup of genes that cause (predominantly murine) CAKUT, if mu- tated, coalesces around the proteoglycan component of the ECM (Supplemental Figure 3, Supplemental Table 3). Proteo- glycans consist of a protein core coupled to unique, variably sulfated glycosamino- glycan chains (Supplemental Figure 3).97,113 Although there are various classes of glycosaminoglycan chains, heparan sul- fate (HS) has previously been implicated to have the largest functional relevance within the developing kidney.113 The synthesis of heparan-sulfate pro- teoglycans (HSPGs) is very complicated and involves a large variety of enzymes (Supplemental Figure 3).114 In contrast, the breakdown is rather simple and pre- dominantly carried out by the enzyme Figure 4. Members of the BMP signaling cascade with a role in CAKUT. Proteins encoded heparanase (HPSE) (Figure 2, Supple- by genes that, if mutated, cause murine CAKUT are outlined in red, and proteins highlighted mental Figure 3).115 The protein in yellow constitute causes of isolated and/or syndromic manifestations of human CAKUT HPSE2 is an inactive enzyme, but it has (Supplemental Table 4). BMPs act as ligands for two classes of transmembrane serine- been reported to function as an endoge- threonine-kinase receptors on the surface of the corresponding effector cells (e.g.,bone 116 morphogenic protein receptor type 1A [BMPR1A] and BMPR2).155 After activated, these nous regulator of HPSE activity. Re- receptors initiate intracellular signaling cascades, including canonical SMAD signaling and cessive mutations in HPSE2 have been fi noncanonical signaling (e.g., via mitogen-activated protein kinase [MAPK]), which even- identi ed in humans and mice with tually result in increased expression of distinct target genes in the cell nucleus.175,176 The urofacial syndrome (OMIM 236730), cellular consequences of BMP signaling depend on the location and local concentration of which includes manifestations from the the BMP ligand.177 The availability of ligands for receptor binding is regulated by diverse CAKUT spectrum within its syndromic intra- and extracellular antagonists and agonists of BMP and includes, for example, phenotype.43,93,100 Gremlin 1 (GREM1), Follistatin (FST), and bone morphogenic protein binding to the en- Overall, the multitude of murine and 178–180 dothelial regulator (BMPER). Please note that, BMPR2, although depicted in the human disease phenotypes that occur as fi gure, has not been implicated in the pathogenesis of CAKUT to date. CRIM1, cysteine- result of disturbances in HSPG biosyn- rich transmembrane bone morphogenic protein regulator 1; CTDNEP1, CTD nuclear en- thesis providesimportant insight into the velope phosphatase 1; GPC3, Glypican 3. mechanisms involved (Supplemental Figure 3, Supplemental Table 3).117,118 Interestingly, the disease-causing mu- redundancy, and an abrogation of any However, although for some HS-relevant tations in FRAS1, FREM1, FREM2,and member of the FC and/or GRIP1 results in enzymes, there are strong loss-of-function GRIP1 (Figure 2) in patients with FS or a failure of the complex to assemble.98,104,107 phenotypes in animal models, mouse Manitoba-oculo-tricho-anal syndrome are A consecutive epithelial-mesenchymal models for other HS-related ECM com- almost exclusively recessive truncating var- detachment is considered causative for ponents (e.g.,mousemodelswitha iants41,42,103,110–112 (Supplemental Figure distinct manifestations of FS (e.g.,skin knockout of HPSE function) do not result 2). Truncating mutations result in an ab- blebbing resulting in cutaneous syndac- in overt defects, thereby suggesting a re- rogation and thereby, loss-of-function of tyly and ).41 dundancy of certain HS enzymes and the corresponding protein. Despite the in- Conversely, we recently found that HSPGs in the ECM that allow for a func- teraction and a structural similarity, mem- missense mutations in FRAS1, FREM1, tional compensation.96,119,120 This hy- bers of the FC do not exhibit functional FREM2,orGRIP1 result in isolated pothesis, however, remains to be validated

42 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 36–50, 2018 www.jasn.org BRIEF REVIEW by additional functional studies in the RAplaysan important role in the tran- phenotype without any extrarenal future. scriptional regulation of developmen- manifestations.140 tally relevant target genes.66,121 Retinoid To the knowledge of the authors, this X Receptor-a (RXR) and Retinoic Acid is the first time that a protein involved in CAKUT AND RA SIGNALING Receptor (RAR; are nuclear receptors RA signaling did not result in a syn- that bind RA each has three subtypes, dromic CAKUT phenotype but rather, Vitamin A is a fat-soluble compound that a, b,andg, with different iso- resulted in a selective CAKUT pheno- is derivedfromdietary sources.121 Vitamin forms).121,141–143 RXR (59)andRAR type. This could potentially point A and its active metabolite, RA,121–123 play (39) assemble to heterodimers and bind toward a role for this coregulator in a important roles in the regulation of a wide to RA-responsive elements in the DNA select group of tissues as opposed to range of biologic processes, including dif- (Figure 3),121,123,141,142,144,145 thereby Retinol Dehydrogenase 10, Aldehyde De- ferentiation, proliferation, and apopto- controlling transcription of functionally hydrogenase 1 Family Member A2, and sis.121,123–130 While exerting essential roles diverse target genes.121,141 The specific Cytochrome P450 Family 26 Subfamily A in adult tissues (e.g., vision, fertility, and subtype and combination of RXRs- Member 1, RARs, and RXRs, which immune response),131–133 vitamin A is RARs thereby most likely permit a cer- exhibit very broad expression patterns. also a well known teratogen.121,123,131,134 tain tissue selectivity, while at the same Although this would have to be further Interestingly, both the deficiency and the time, sustaining the pleiotropy of RA- elucidated, the possibilityexiststhat, in the excess of vitamin A during pregnancy mediated effects.123 RXR and RAR can future, more regulators of RA-dependent cause similar forms of an embryopathy additionally bind coregulators (coactiva- transcriptional regulation may be identi- known as vitamin A–deficient (VAD) syn- tors and corepressors), which adds fied in patients with cases with isolated drome.121,131,135 VAD syndrome affects another level of complexity to the tran- features from within the syndromic VAD many structures, including the ocular, car- scriptional regulation of RA-dependent phenotype. diac, respiratory, and urinary systems, and genes in an RA-dependent man- NRIP1 mutations as a cause of is a cause of CAKUT.123,135,136 It is, there- ner.121,131,141,142,146–149 A potential con- CAKUT are of particular interest, be- fore, recommended to carefully monitor tribution to the tissue specificity of cause they represent the first form of vitamin A levels during pregnancy.121 RA-mediated effects via tissue-specific CAKUT that could be amenable to pre- Over the past decades,efforts, mainly led expression patterns of coregulators is vention treatment. It will have to be tested by Mendelsohn et al.,66,122,130,137–139 have likely. whether control of RA metabolism may broadened the understanding of the molec- Mutations in genes encoding enzymes be applicable in pregnant women with a ular mechanisms that underlie the well that are involved in intracellular process- family history of NRIP1-related cases of established interplay of vitamin A/RA sig- ing of RA, such as Retinol Dehydroge- CAKUT. NRIP1 mutations as genetic naling and renal morphogenesis in mice. nase 10,150 Aldehyde Dehydrogenase 1 cause of human CAKUT are very rare, Research by Chia et al.66 and Batourina Family Member A2,138 and Cytochrome however, with only one case of familial et al.122 (among others) convincingly P450 Family 26 Subfamily A Member CAKUT to date.140 showed that RA is required for two key 1,151,152 as well as double mutants of spe- events during embryonic development of cific isoforms within the RXR (Rxra) the urinary tract: the insertion of the neph- and RAR family of proteins (Rara and CAKUT AND SIGNALING ric duct into the cloaca (approximately Rarb)123 (Figure 3) have previously been THROUGH BMPS E9.5) (Figure 1B) as well as the branching shown to result in multisystemic pheno- morphogenesis of the UB (approximately typesinmicethatresembleVADsyn- Bone morphogenic proteins (BMPs) E11.5–E15.5) (Figure 1, D and E). In both drome and present manifestations from constitute the largest family within the scenarios, the mechanism by which vitamin within the CAKUT spectrum. TGFb superfamily of molecules.155 A/RA mainly exerts its effects seems to be NRIP1 is a member of a group of BMPs were originally identified as due to control of expression levels of a key ligand-inducible transcription factors, bone-derived proteins capable of induc- player of renal morphogenesis, Ret.66,122 and it is involved in the coregulation of ing ectopic bone formation in vivo.156,157 Although the pathophysiologic rele- variety of biologic processes (e.g., estro- Nowadays,theBMPpathwayisre- vance of vitamin A for embryologic de- gen and thyroid hormone signaling), in- nowned for its importance in general velopment, including processes of renal cluding RA-mediated pathways.153,154 embryogenesis, with diverse roles de- morphogenesis, has long been known, NRIP1 has been shown to have the ca- pending on the spatial and temporal very recently for the first time, a hetero- pacity to interact with both RAR and context.78 Alargenumberofin vitro zygous mutation in a gene related to RXR (Figure 3).153 Despite the large va- and in vivo studies underscore a crucial vitamin A function (Nuclear Receptor In- riety of biologic functions of NRIP1, in role of BMP signaling for renal morpho- teracting Protein 1 [NRIP1]) was discov- the examined family with seven af- genesis in animal models (Figure 4, Sup- ered in one large kindred with isolated fected individuals, the identified muta- plemental Table 4).79–92,158–160 Also, the human CAKUT.140 tion resulted in an isolated CAKUT development of the human kidney and

J Am Soc Nephrol 29: 36–50, 2018 Monogenic CAKUT 43 BRIEF REVIEW www.jasn.org urinary tract seems to depend on intact In general, BMP ligands induce cellu- lower than that in other causes of early- BMP signaling. This is indicated by the lar responses by forming complexes with onset CKD, being approximately 30% in finding of mutations in genes inter-related two major types of diverse membrane- steroid-resistant nephrotic syndrome181 with BMP signaling in patients with iso- bound serine-threonine kinase recep- and 70% in renal cystic diseases.182 lated (BMP421 and GREM142)orsyndromic tors: type 1 BMP receptors and type 2 Monogenic CAKUT genes are more dif- (BMPER,94 BMP4,95 and GPC3161)mani- BMP receptors.155 Both types 1 and 2 ficult to identify due to the high contri- festations of human CAKUT. receptors are required for subsequent bution of autosomal dominant renal Details of the importance of BMP transduction of the BMP signal.173,174 developmental genes, which often ex- signaling for renal morphogenesis The cellular responses, however, seem hibit features of incomplete penetrance have been previously reviewed in de- to be defined by the type 1 receptor.155 (lack of genotype-phenotype correla- tail78,155,162–164 and are continually be- Downstream of ligand-receptor binding, tion) and variable expressivity (differing ing revealed. Experimental evidence BMPs activate either a canonical or a organ involvement). Previous experi- suggests that the contribution of BMPs noncanonical signaling cascade through ence, however, strongly suggests that, to renal morphogenesis is very complex. of downstream effec- before complex genetic mechanisms Research in the field of BMP signaling is tors. The canonical BMP pathway trans- have to be evoked, such as polygenic in- generally challenged by the early embry- mits signals via Smad proteins.175,176 heritance or epigenetic mechanisms, onic lethality that often results from Noncanonical BMP signaling includes hundreds of monogenic causative genes loss-of-function mutations in animal the activation of the mitogen-activated for CAKUT genes will still be identified. models of BMP-related genes (Supple- protein kinase family of signaling mole- In this context, the ability to represent mental Table 4). This complicates the cules.175 Both signaling cascades eventu- apatient’s exact mutation in an animal investigation of respective contributions ally result in the increased transcription model of CAKUT (e.g.,miceorXenopus) to the development of the kidneys and of corresponding target genes in the using CRISPR/Cas knockin techniques – the urinary tract.85,165 167 Spatiotempo- nucleus. The location and local concen- has strong potential to conclusively de- ral differences in expression patterns of tration of the BMP ligand hereby deter- fine genotype-phenotype relations for different components of the BMP sig- mine the exact cellular consequences of patients with CAKUT. naling cascade indicate that proteins BMP signaling.177 Intra- and extracellular Identification of monogenic causes of from the BMP family perform various agonists (e.g., BMPER) and antagonists CAKUT by WES will rapidly become functions for both UB and mesenchy- (e.g., GREM1 and FST) of BMP tightly common practice in pediatric nephrol- mal cells during development of the regulate the level of ligand-receptor inter- ogy and urology clinics due to reduction – murine kidney.79,84,89,168 171 Tight reg- action at the ligand or (more rarely) the in cost for WES and continuous im- – ulation of these proteins seems to be receptor level,178 180 and they play an im- provement of algorithms that define critical in these processes. This is, for portant role in renal development. deleteriousness of mutant alleles. fi fi instance, exempli ed by the nding of Discovery of additional monogenic CAKUT phenotypes resulting from CAKUT genes will strongly aide our un- bothmicewithnullmutationsindis- CONCLUSION derstanding of the complex pathogenesis tinctBMPgenesaswellasmicewitha of CAKUT. In addition, molecular genet- lack of inhibition of the respective In conclusion, a long-standing hypothe- ic diagnostics using WES may offer im- 78,84 fi BMP. However, it was shown that sis has recently found strong con rma- portant advances to clinical management BMPs may be able to substitute one an- tion; it stated that CAKUT, to a large part, for patients with CAKUT, who represent other during murine renal morphogen- is caused by single-gene mutations in a about 50% of all patients with ESRD be- esis as previously shown for Bmp7 and high number of different monogenic fore age 25 years old, because causative 172 Bmp4. CAKUT genes, involving different genes mutations will soon allow for prognostic Mechanisms of BMP signaling in in different patients. conclusions, such as outcomes of recon- the context of renal development are Whole-exome sequencing (WES) has structive surgery, rate of progression into briefly outlined in Figure 4. Figure 4 facilitated discovery of these genes. Even fi renal failure, and the likelihood for the depicts components of the signaling cas- the subhypotheses found con rmation, development of extrarenal complications. cade that have previously been reported to stating that monogenic CAKUT genes result in manifestations of murine would often represent candidate genes CAKUT if mutated (outlined in red in Fig- from mouse models of CAKUT, renal de- ure 4).79–92,158–160 Some of these genes/ velopmental genes, or genes known to ACKNOWLEDGMENTS proteins have additionally been identified cause human clinical syndromes that in human forms of isolated (BMP421 and have a CAKUT component. A.T.v.d.V. is supported by postdoctoral re- GREM142)orsyndromic(BMPER,94 The fraction of approximately 17% of search fellowship VE916/1-1 from the German GPC3,161 and BMP495) CAKUT (high- patients with CAKUT, in whom a mono- Research Foundation (DFG). A.V.is supported lighted in yellow in Figure 4). genic cause can be detected by WES, is by a Fulbright postdoctoral scholar award and

44 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 36–50, 2018 www.jasn.org BRIEF REVIEW grants from the Manton Center Fellowship Kidney and Urinary Tract, edited by Barakat urinary tract (CAKUT). Nephrol Dial Trans- – Program, Boston Children’s Hospital, and the A, Rushton H, Cham, Switzerland, Springer plant 27: 2355 2364, 2012 Nature, 2016, pp 303–322 23. Lopez-Rivera E, Liu YP, Verbitsky M, Mallinckrodt Research Fellowship. F.H. is the 11. Vivante A, Kohl S, Hwang DY, Dworschak Anderson BR, Capone VP, Otto EA, Yan Z, William E. Harmon Professor of Pediatrics. GC, Hildebrandt F: Single-gene causes of Mitrotti A, Martino J, Steers NJ, Fasel DA, This work was supported by National Institutes congenital anomalies of the kidney and Vukojevic K, Deng R, Racedo SE, Liu Q, of Health grant DK088767 (to F.H.). F.H. is urinary tract (CAKUT) in humans. 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50 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 36–50, 2018 a b c

d e

Supplementary Figure 1. Stages of nephron development. a) Metanephric mesenchyme in closest proximity to the ureteric bud condenses around the ureteric bud-tips and forms the cap mesenchyme. b) The cap mesenchyme subsequently gives rise to the most primitive nephron progenitor structure, the epithelial vesicle. During the maturation process, the epithelial vesicle sequentially undergoes distinct developmental stages, i.e. c) the comma-shaped body, d) the S-shaped body, and e) the mature nephron.

(From Kamath, Spinner & Rosenblum, Nat Rev Neph, 2013).

This hypothesis will have to further be tested by the generation of additional genetic and and genetic additional of the future. generation in data functional the by tested be further to have will hypothesis This milder FREM2 be to “ an found indicating thus phenotype were CAKUT isolated with (“ patients in identified Mutations genes. CAKUT MOTA/BNAR syndrome isolated and Fraser- known these predominantly in mutations recessive carry with phenotypes individuals of ~2.2%) (13/590; (Kohl proportion significant al. et Kohl by Studies ( anomalies) renal and (Manitoba- syndrome MOTA/BNAR of ( syndrome Fraser of phenotypes CAKUT syndromic severe, FREM2) FREM1, Biallelic Fraser-complex. the encoding genes in mutations of variability Phenotypic 2. Figure Supplementary hypomorphic Severity Biallelic previously reported , truncating mutations in genes encoding members of the Fraser-Complex ( Fraser-Complex the of members encoding genes in mutations truncating GRIP1 ”) than mutations published to be causative of the respective syndromic syndromic respective the of causative be to published mutations than ”) and and the associated protein protein associated the and truncating FREM1 FREM1 genes. genes. ). Biallelic allelism JASN JASN mutations oculo ” for the broad phenotypic spectrum of the the of spectrum phenotypic broad the for ” 25:1917-22, 2014), however revealed that also a a also that revealed however 2014), 25:1917-22, - tricho missense GRIP1 -anal/bifid nose with or without anorectal anorectal without or with nose -anal/bifid are known monogenic causes of the the of causes monogenic known are mutations FRAS1, FREM2, GRIP1 FREM2, FRAS1, FRAS1 FRAS1, ) and and ) , Ndst1 Glce Hs2st1 Heparanase 2 Sulfatase 1 & HPSE2 (inactive) Sulfatase 2 Sulf1&2

cleavage Heparanase

Membrane- Transmembrane Secreted bound (e.g. HSPG (e.g. perlecan/ glypican) HSPG2) Glypican 3 GPC3

Supplementary Figure 3. Overview of enzymes involved in synthesis and breakdown of heparan-sulfate proteoglycan (HSPG) chains.

The synthesis of HSPG requires a multiplicity of different enzymatic reactions. The genes encoding enzymes in the HSPG pathway Ndst1, Glce, Hs2st1, Sulf1 & Sulf2, HPSE2 as well as one HSPG subtype GPC3 have previously been implicated to cause syndromic CAKUT phenotypes, if mutated in mice (blue). HPSE2 and GPC3 are also known to cause CAKUT in humans, if mutated (red) (see also Supplementary Table 1). The role of ECM proteins in renal morphogenesis and the pathogenesis of CAKUT is related to their importance for structural integrity and/or regulation of the intricate cross-talk between the structures of the developing kidney. (modified from Lander, Selleck, J Cell Biol 148:227, 2000).

Supplementary Table 1: 183 Monogenic causes of murine CAKUT. 183 genes that, if mutated, cause CAKUT in mice were retrieved from the MGI database (http://www.informatics.jax.org/) by searching for the following terms: “abnormal mesonephric mesenchyme”, “abnormal mesonephric mesenchyme morphology”, “abnormal metanephric mesenchyme”, “abnormal metanephric mesenchyme morphology”, “abnormal metanephric ureteric bud development”, “abnormal ureter development”, “abnormal ureter morphology”, “abnormal ureteric bud elongation”, “abnormal ureteric bud invasion”, “abnormal ureterovesical junction”, “abnormal urinary system development”, “absent kidney”, “absent metanephric mesenchyme”, “absent metanephros”, “absent ureter”, “dilated ureter”, “double kidney pelvis”, “double kidney pelvis”, “double ureter”, “duplex kidney”, “ectopic ureter”, “ectopic ureteric bud”, “hydroureter”, “impaired branching involved in ureteric bud morphogenesis”, “pelvic kidney”, “renal hypoplasia”, “short ureter”, “single kidney”, “small metanephros”, “ureter hypoplasia”, “abnormal nephrogenic mesenchyme morphogenesis”, “ureteropelvic junction obstruction”. Mouse CAKUT genes are listed here in parallel with the corresponding human phenotype that was extracted from the OMIM database (www.omim.org). Human phenotypes representing CAKUT are underlined (source: clinical synopsis table, OMIM, www.omim.org). Human PubMed Human Phenotype Gene Protein ID MGI phenotype (contains Ref. ID OMIM # CAKUT) Renal tubular

Ace Angiotensin I converting enzyme 8642790 267430 dysgenesis Heterotaxy,

Acvr2b Activin A Receptor Type 2B 9242489 visceral, 4, 613751 autosomal

ADAM Metallopeptidase With

Adamts1 10811842 - - Thrombospondin Type 1 Motif 1

Renal tubular Agt Angiotensinogen 8675666 267430 dysgenesis Renal tubular

Agtr1a Angiotensin II receptor, type 1a 10024874 267430 dysgenesis Renal tubular

Agtr1b Angiotensin II receptor, type 1b 10024874 267430 dysgenesis

Agtr2 Angiotensin II Receptor Type 2 10024874 - -

Aldehyde Dehydrogenase 1

Aldh1a2 20040494 - - Family Member A2

Osteopathia striata APC Membrane Recruitment

Amer1 21571217 with cranial 300373 Protein 1 sclerosis Acidic Nuclear Phosphoprotein

Anp32b 21636789 - - 32 Family Member B

Adenine Adenine phosphoribosyl-

Aprt 8864750 614723 Phosphoribosyltransferase transferase deficiency Diabetes insipidus,

Aqp2 Aquaporin 2 3184310 125800 nephrogenic

Arhgap1 Rho GTPase Activating Protein 1 17227869 - -

Arhgap3 Rho GTPase Activating Protein

26859289 - - 5 35

Arid5b AT-Rich Interaction Domain 5B 17143286 - -

ADP Ribosylation Factor Like

Arl3 16565502 - - GTPase 3

Atmin ATM Interactor 24852369 - - Menkes disease; Occipital horn 309400; ATPase Copper Transporting

Atp7a 11534785 syndrome; Spinal 304150; Alpha

muscular atrophy, 300489 distal, X-linked 3 Hepatocellular 17246824

Axin1 Axin 1 carcinoma, 114550

13340237 somatic

Bag6 BCL2 Associated Athanogene 6 16287848 - - Leukemia/

Bcl2 BCL2, Apoptosis Regulator 8623928 lymphoma, B-cell, n/a 2 ,

Bmp4 Bone Morphogenetic Protein 4 10749566 607932 syndromic 6

Bmp5 Bone Morphogenetic Protein 5 5692092 - -

Bmp7 Bone Morphogenetic Protein 7 7590254 - - Diaphano- BMP Binding Endothelial

Bmper 17035289 spondylo- 608022 Regulator dysostosis COACH syndrome; Joubert 216360; Coiled-Coil And C2 Domain Cc2d2a J:175213 syndrome 9; 612285; Containing 2A

Meckel syndrome 612284 6 Takenouchi-

Cdc42 Cell Division Cycle 42 23555292 616737 Kosaki syndrome

Cdh4 Cadherin 4 11839813 - -

Cdh6 Cadherin 6 10864459 - - Cholinergic Receptor Muscarinic Prune belly

Chrm3 10944224 100100 3 syndrome Cntrl Centriolin J:175213 - -

Crumbs 3, Cell Polarity Complex

Crb3 26631503 - - Component

Cysteine Rich Transmembrane

Crim1 22511315 - - BMP Regulator 1

CTD Nuclear Envelope

Ctdnep1 23360989 - - Phosphatase 1

Colorectal cancer, somatic; Hepatocellular carcinoma, 114500; somatic; Medullo- 114550; blastoma, somatic; 155255;

Ctnnb1 Catenin Beta 1 20454682 Mental retardation, 615075; autosomal 167000; dominant 19;

132600 Ovarian cancer, somatic; Pilomatricoma, somatic Catenin Beta Interacting Protein

Ctnnbip1 17803964 - - 1

C-X-C Motif Chemokine Receptor

Cxcr4 J:175213 WHIM syndrome 193670 4

Cytochrome P450 Family 26

Cyp26a1 11157778 - - Subfamily A Member 1

Dishevelled Binding Antagonist

Dact1 20145239 - - Of Beta Catenin 1

Mitral valve prolapse 2; Van 607829;

Dchs1 Dachsous Cadherin-Related 1 21303848

Maldergem 601390 syndrome 1 Smith-Lemli-Opitz

Dhcr7 7-Dehydrocholesterol Reductase 11230174 270400 syndrome Discs Large MAGUK Scaffold

Dlg1 17172448 - - Protein 1 Discs Large MAGUK Scaffold {Inflammatory

Dlg5 17765678 612288 Protein 5 bowel disease 20} Ciliary dyskinesia, Dynein Axonemal Heavy Chain primary, 7, with or

Dnah11 J:175213 611884 11 without situs inversus Ciliary dyskinesia, primary, 3, with or

Dnah5 Dynein Axonemal Heavy Chain 5 J:175213 608644 without situs inversus Dyggve-Melchior- Clausen disease; 223800;

Dym Dymeclin 18852472

Smith-McCort 607326 dysplasia Short-rib thoracic Dynein Cytoplasmic 2 Heavy

Dync2h1 J:175213 dysplasia 3 with or 613091 Chain 1 without polydactyly

Efnb2 Ephrin B2 15223334 - -

Emx2 Empty Spiracles Homeobox 2 9165114 Schizencephaly 269160

Estrogen Related Receptor

Esrrg 21138943 - - Gamma

Early transposon element Epilepsy, familial

Etl4/Etn2 23436999 611631 insertion site 2 temporal lobe, 4

Etv4 ETS variant 4 19898483 - -

Etv5 ETS variant 5 19898483 - -

Exoc5 Exocyst complex component 5 26046524 - - Branchiooto-renal syndrome 1, with or without cataracts; 113650; Branchiootic EYA Transcriptional Coactivator 602588;

Eya1 10471511 syndrome 1; And Phosphatase 1 602588; Anterior segment

166780 anomalies with or without cataract; Otofaciocervical syndrome Van Maldergem syndrome 2; Hennekam lymph- 615546;

Fat4 FAT Atypical Cadherin 4 21303848

angiectasia- 616006 lymphedema syndrome 2 Aplasia of lacrimal and salivary 180920;

Fgf10 Fibroblast Growth Factor 10 11062007

glands; LADD 149730 syndrome

Fgf7 Fibroblast Growth Factor 7 9876183 - - Hypo-gonadotropic hypogonadism 6

Fgf8 Fibroblast Growth Factor 8 16049111 612702 with or without anosmia Antley-Bixler syndrome; ; Beare- Stevenson cutis gyrata syndrome; Bent bone dysplasia 207410; syndrome; 101200; Craniofacial- 123790; skeletal- 614592; dermatologic 101600; dysplasia; Fibroblast Growth Factor 123500;

Fgfr2 15843416 Crouzon Receptor 2 613659; syndrome; Gastric 123150; cancer, somatic; 149730; Jackson-Weiss 101600; syndrome; LADD 101400; syndrome; Pfeiffer

609579 syndrome; Saethre-Chotzen syndrome; Scaphocephaly, maxillary retrusion, and mental retardation

Fibroblast Growth Factor

Fgfrl1 19715689 - - Receptor-Like 1

Fmn1 Formin 1 7517224 - - Anterior segment dysgenesis 3, 601631;

Foxc1 5500588 multiple subtypes;

602482 Axenfeld-Rieger syndrome, type 3

Foxd1 8666231 - -

Foxd2 Forkhead Box D2 10648626 - - Rett syndrome,

Foxg1 Forkhead Box G1 16109771 613454 congenital variant Fraser Extracellular Matrix

Fras1 12766769 Fraser syndrome 219000 Complex Subunit 1

Bifid nose with or without anorectal and renal 608980; FRAS1 Related Extracellular anomalies;

Frem1 12766769 248450; Matrix 1 Manitoba

614485 oculotrichoanal syndrome; Trigonocephaly 2

FRAS1 Related Extracellular

Frem2 12766769 Fraser syndrome 219000 Matrix Protein 2

Fstl1 Follistatin Like 1 22485132 - - Exudative vitreoretinopathy 133780;

Fzd4 Frizzled Class Receptor 4 21343368

1; Retinopathy of 133780 prematurity

Fzd8 Frizzled Class Receptor 8 21343368 - - Emberger syndrome; 614038;

Gata2 GATA Binding Protein 2 18233958

Immunodeficiency 614172 21 Hypopara- thyroidism,

Gata3 GATA Binding Protein 3 16319112 sensorineural 146255 deafness, and renal dysplasia

Gdf11 Growth Differentiation Factor 11 12729564 - - Central Glial Cell Derived Neurotrophic

Gdnf 11422733 hypoventilation 209880 Factor syndrome

Gfra1 GDNF Family Receptor Alpha 1 23542432 - -

Glce Glucuronic Acid Epimerase 12788935 - - Greig cephalopoly- syndactyly syndrome; 175700; Pallister-Hall 146510;

Gli3 GLI Family 3 11978771 syndrome; 174200; Polydactyly,

174700 postaxial, types A1 and B; Polydactyly, preaxial, type IV

Simpson-Golabi- Behmel syndrome, 312870;

Gpc3 Glypican 3 10402475

type 1; Wilms 194070 tumor, somatic

Gremlin 1, DAN Family BMP

Grem1 15201225 - - Antagonist

Glutamate Receptor Interacting

Grip1 10974668 Fraser syndrome 219000 Protein 1 Diabetes mellitus, noninsulin- 125853;

Hnf1b HNF1 Homeobox B 23362348 dependent; Renal

137920 cysts and diabetes syndrome Radioulnar synostosis with

Hoxa11 Homeobox A11 7596412 amegakaryo-cytic 605432 thrombocyto-penia 1

Hoxd11 Homeobox D11 12050119 - - Guttmacher syndrome; Hand- 176305;

Hoxa13 Homeobox A13 12783783

foot-uterus 140000 syndrome

Hoxc10 Homeobox C10 19623272 - -

Hoxc11 Homeobox C11 12050119 - - Urofacial

Hpse2 Heparanase 2 25510506 236730 syndrome 1 Heparan Sulfate 2-O-

Hs2st1 9637690 - - Sulfotransferase 1

Hydroxysteroid 17-Beta

Hsd17b2 18048640 - - Dehydrogenase 2

Heat Shock Protein Family A

Hspa4l 16923965 - - (Hsp70) Member 4 Like

5-Hydroxytryptamine Receptor

Htr3a 15201326 - - 3A Inhibitor Of DNA Binding 2, HLH

Id2 15569159 - - Protein

Ilk Integrin Linked Kinase 19829382 - - Interstitial lung disease, nephrotic

Itga3 Integrin Subunit Alpha 3 10433923 syndrome, and 614748 epidermolysis bullosa, congenital Epidermolysis bullosa, junctional,

Itga6 Integrin Subunit Alpha 6 10433923 226730 with pyloric stenosis Renal 9054500

Itga8 Integrin Subunit Alpha 8 hypodysplasia/ 191830

17537792 aplasia 1

Itgb1 Integrin Subunit Beta 1 19439520 - -

Kif26b Kinesin Family Member 26B 20439720 - -

Lama5 Laminin Subunit Alpha 5 10625553 - -

Lamc1 Laminin Subunit Gamma 1 12015298 - -

{Bone mineral Leucine Rich Repeat Containing 21523854

Lgr4 density, low, 615311

G Protein-Coupled Receptor 4 22738954 susceptibility to}

Luteinizing Hormone/Choriogona

Lhx1 16216236 - - do-tropin Receptor

Lin-7 Homolog C, Crumbs Cell

Lin7c 17923534 - - Polarity Complex Component

Sclerosteosis 2; Cenani-Lenz syndactyly 614305;

Lrp4 LDL Receptor Related Protein 4 20454682 syndrome; 212780;

?Myasthenic 616304 syndrome, congenital, 17 Tumor

Lzts2 21949185 - - Suppressor 2 Carpenter

Megf8 Multiple EGF Like Domains 8 18043505 614976 syndrome 2 Meckel syndrome

Mks1 Meckel Syndrome, Type 1 21045211 249000 1 ?Winchester

Mmp14 Matrix Metallopeptidase 14 20727881 277950 syndrome

Mmp17 Matrix Metallopeptidase 17 21347258 - -

V- Avian Myelocytomatosis Feingold

Mycn Viral Oncogene Neuroblastoma 1459449 164280 syndrome 1 Derived Homolog

Mental retardation, N-Deacetylase And N-

Ndst1 autosomal 616116 Sulfotransferase 1 recessive 46 Neurofibromatosis,

Nf1 Neurofibromin 1 7926784 162200 type 1

Nfia A 17530927 - -

Nicotinamide Nucleotide

Nmnat2 23082226 - - Adenylyltransferase 2

Brachydactyly, type B2; Multiple synostoses syndrome 1; 611377; Stapes ankylosis 186500;

Nog Noggin 18028901 with broad thumb 184460; and toes; 185800;

Symphalangism, 186570 proximal, 1A; Tarsal-carpal coalition syndrome Alagille syndrome 610205;

Notch2 Notch 2 20299358 2; Hajdu-Cheney

102500 syndrome

Npnt Nephronectin 17537792 - - Odd-Skipped Related

Osr1 16790474 - - Transciption Factor 1

Parva Parvin Alpha 19829382 - - Papillorenal syndrome; 120330;

Pax2 Paired Box 2 8575306 Glomerulo-

616002 sclerosis, focal segmental, 7 Hypothyroidism, congenital, due to

Pax8 Paired Box 8 12435636 218700 thyroid dysgenesis or hypoplasia Leukemia, acute

Pbx1 PBX Homeobox 1 12591246 176310 pre-B-cell Microcephalic osteodysplastic

Pcnt Pericentrin (kendrin) 25220058 210720 primordial dwarfism, type II

Proprotein Convertase

Pcsk5 18519639 - - Subtilisin/Kexin Type 5

Gastrointestinal stromal tumor, somatic; Hyper- Platelet Derived Growth Factor eosinophilic 606764;

Pdgfra 19217431

Receptor Alpha syndrome, 607685 idiopathic, resistant to imatinib PDS5 Cohesin Associated Factor

Pds5a 19412548 - - A

Plxnb1 Plexin B1 18799546 - -

Plxnb2 Plexin B2 21035938 - - Plxnd1 Plexin D1 J:175213 - -

Protein Phosphatase 3

Ppp3r1 15057312 - - Regulatory Subunit B, Alpha

Epilepsy, Prickle Planar Cell Polarity

Prickle1 25190059 progressive 612437 Protein 1 myoclonic 1B Basal cell carcinoma, 605462; somatic; Basal cell

Ptch1 Patched 1 22792366 109400; nevus syndrome;

610828 Holopros- encephaly 7 Bannayan-Riley- Ruvalcaba syndrome; Cowden syndrome 1; Endometrial carcinoma, somatic; 153480; Lhermitte-Duclos 158350; syndrome; 608089; Macrocephaly/auti Phosphatase And Tensin 158350;

Pten 17540362 sm syndrome; Homolog 605309; Malignant 155600; melanoma, 275355; somatic;

276950 Squamous cell carcinoma, head and neck, somatic; VATER association with macrocephaly and ventriculo-megaly ?Breasts and/or Protein Tyrosine Phosphatase,

Ptprf 19273906 nipples, aplasia or 616001 Receptor Type F hypoplasia of, 2

Pygo1 Pygopus Family PHD Finger 1 17425782 - -

Pygo2 Pygopus Family PHD Finger 2 17425782 - -

Leukemia, acute

Rara Alpha 9376317 612376 promyelocytic

Retinol Dehydrogenase 10 (All- 21930923 Rdh10 - -

Trans) 17473173

Neurodevelopment al disorder with or Arginine-Glutamic Acid Dipeptide

Rere 23451234 without anomalies 616975 Repeats of the brain, eye, or heart Central hypoventilation syndrome, congenital; 209880; Medullary thyroid 155240;

Ret Ret Proto-Oncogene 16452504 carcinoma; 171400; Multiple endocrine 162300;

neoplasia IIA; 171300 Multiple endocrine neoplasia IIB; Pheochromo- cytoma

Roundabout Guidance Receptor Robo1 J:175213 - - 1 Roundabout Guidance Receptor Vesicoureteral

Robo2 17357069 610878 2 reflux 2 17904116 Rspo2 R-Spondin 2 - -

12782276 Townes-Brocks syndrome;

Sall1 Spalt Like Transcription Factor 1 11688560 Townes-Brocks 107480 branchio-otorenal- like syndrome Duane-radial ray 607323;

Sall4 Spalt Like Transcription Factor 4 17216607 syndrome; IVIC

147750 syndrome

Sc5d Sterol-C5-Desaturase J:175213 Lathosterolosis 607330 Epilepsy, progressive Scavenger Receptor Class B

Scarb2 12620969 myoclonic 4, with 254900 Member 2 or without renal failure {Hypogonado- tropic

Sema3a Semaphorin 3A 18249526 hypogonadism 16 614897 with or without anosmia} SEC14 And Spectrin Domain

Sestd1 23696638 - - Containing 1 Holopros- encephaly 3; Microphthalmia 142945; with 5; 611638;

Shh Sonic Hedgehog 12399320 Schizencephaly; 269160; Single median 147250 maxillary central incisor Branchiootic syndrome 3; 608389;

Six1 SIX Homeobox 1 14695375 Deafness, 605192 autosomal dominant 23

Six2 SIX Homeobox 2 17036046 - -

Slit2 Slit Guidance Ligand 2 15130495 - -

Slit3 Slit Guidance Ligand 3 14550534 - -

Sox4 SRY-Box 4 16109771 - - Acampomelic campomelic dysplasia; Campomelic

Sox9 SRY-Box 9 20881014 dysplasia; 114290 Campomelic dysplasia with autosomal sex reversal

Sprouty RTK Signaling

Spry1 15691764 - - Antagonist 1

Sulf1 Sulfatase 1/ Sulfatase 2 17593974 - -

Sulf2 Sulfatase 1/ Sulfatase 2 17593974 - -

Tbc1d32 TBC1 Domain Family Member 32 J:175213 - - Congenital anomalies of

Tbx18 T-Box 18 24016759 143400 kidney and urinary tract 2 Spondylocostal

Tbx6 T-Box 6 4073528 122600 dysostosis 5

Tcf21 Transcription Factor 21 10572052 - -

Tfcp2l1 Transcription Factor CP2-Like 1 17079272 - -

Transforming Growth Factor Beta Loeys-Dietz

Tgfb2 9217007 614816 2 syndrome 4 Adrenal cortical carcinoma; Breast cancer; Choroid 202300; plexus papilloma; 114480; Colorectal cancer; 260500; Hepatocellular 114500;

Trp53 Transformation related protein 53 11780111 carcinoma; Li- 114550; Fraumeni 151623; syndrome; 607107; Nasopharyngeal 259500;

carcinoma; 260350 Osteosarcoma; Pancreatic cancer Trichorhino- phalangeal Transcriptional Repressor GATA syndrome, type I; 190350;

Trps1 19820125

Binding 1 Trichorhino- 190351 phalangeal syndrome, type III

Tshz3 Teashirt Zinc Finger Homeobox 3 18776146 - -

Tyr Tyrosinase J:179802 - -

Upk3a Uroplakin 3A 11085999 - -

WAS/WASL Interacting Protein

Wasl 23555292 - - Family Member 1 ?Congenital heart defects, WD Repeat Containing Planar

Wdpcp 24302887 hamartomas of 217085 Cell Polarity Effector tongue, and polysyndactyly

Wnt11 Wnt Family Member 11 12783789 - -

Mullerian aplasia and hyperandro- 158330;

Wnt4 Wnt Family Member 4 7990960

genism; ?SERKAL 611812 syndrome ,

Wnt5a Wnt Family Member 5A J:175213 180700 autosomal dominant 1

Wnt7b Wnt Family Member 7B 19060336 - -

Wnt9b Wnt Family Member 9B 16054034 - - Denys-Drash syndrome; Frasier syndrome; 194080; Meacham 136680; syndrome; 608978;

Wt1 Wilms Tumor 1 18040647 Mesothelioma, 156240; somatic; Nephrotic 256370;

syndrome, type 4; 194070 Wilms tumor, type 1

Orofaciodigital

Xpl X-linked polydactyly 7391545 311200 syndrome I

Coloboma, ocular; Coloboma, ocular, with or without

Yap1 Yes Associated Protein 1 23555292 hearing 120433 impairment, cleft lip/palate, and/or mental retardation

Zinc Finger And BTB Domain Zbtb14 J:175213 - - Containing 14

Supplementary Table 2: Human monogenic CAKUT genes with reported involvement in GDNF-RET signaling. List of human CAKUT genes with known (direct or indirect) involvement in GDNF-RET signaling (upstream or downstream of RET). Please note that genes in parenthesis have not been explicitly mentioned in the references provided themselves, but are part of pathways that otherwise have been demonstrated to be involved.

Gene Protein Reference Autosomal dominant Woolf and Davies JASN 24:19 2013; Short and Smyth Nat Rev Nephr 12:754 2016; Schedl Nat Rev BMP4 Bone morphogenic protein 4 Genet 8:791 2007; Davis Pediatr Nephrol 29:597 2014 Short and Smyth Nat Rev Nephr 12:754 2016; EYA1 Eyes absent homolog 1 Schedl, Nat Rev Genet 2007; Davis Pediatr Nephrol 29:597 2014 Costantini Dev Biol 1:693 2012; Davis Pediatr GATA3 GATA binding protein 3 Nephrol 29:597 2014 HNF1B HNF homeobox B Costantini Dev Biol 1:693 2012 Nuclear Receptor Interacting Costantini Dev Biol 1:693 2012; Davis Pediatr (NRIP1) Protein 1 Nephrol 29:597 2014 Short and Smyth Nat Rev Nephr 12:754 2016; PAX2 Paired box 2 Schedl Nat Rev Genet 8:791 2007; Costantini Dev Biol 1:693 2012; Davis Pediatr Nephrol 29:597 2014 Woolf and Davies JASN 24:19 2013; Short and Proto-oncogene tyrosine- Smyth Nat Rev Nephr 12:754 2016; Schedl Nat Rev RET protein kinase receptor Ret Genet 8:791 2007; Costantini Dev Biol 1:693 2012; Davis Pediatr Nephrol 29:597 2014 Roundabout, axon guidance Short and Smyth Nat Rev Nephr 12:754 2016; ROBO2 receptor, homolog 2 Schedl Nat Rev Genet 8:791 2007; Davis Pediatr (Drosophila) Nephrol 29:597 2014 Sal-like protein 1 (also known Short and Smith Nature Reviews, 2016; Schedl, Nat SALL1 as spalt-like transcription Rev Genet, 2007; Davis Pediatr Nephrol 29:597 2014 factor 1) Costantini Dev Biol 1:693 2012; Davis Pediatr SIX1 SIX homeobox 1 Nephrol 29:597 2014 SIX2 SIX homeobox 2 Short and Smith, Nature Reviews, 2016 Short and Smyth Nat Rev Nephr 12:754 2016; SLIT2 Slit homolog 2 Schedl Nat Rev Genet 8:791 2007; Davis Pediatr Nephrol 29:597 2014 Short and Smyth Nat Rev Nephr 12:754 2016; SLIT-ROBO Rho GTPase (SRGAP1) Schedl Nat Rev Genet 8:791 2007; Davis Pediatr activating protein 1 Nephrol 29:597 2014 Autosomal recessive Angiotensin-converting Woolf and Davies JASN 24:19 2013; Davis Pediatr (ACE) enzyme Nephrol 29:597 2014 Woolf and Davies JASN 24:19 2013; Davis Pediatr (AGT) Angiotensinogen Nephrol 29:597 2014 AGTR1 Angiotensin II receptor, type 1 Davis Pediatr Nephrol 29:597 2014 Extracellular matrix protein FRAS1 Short and Smyth Nat Rev Nephr 12:754 2016 FRAS1 FRAS1 related extracellular FREM1 Short and Smyth Nat Rev Nephr 12:754 2016 matrix protein 1 FRAS1 related extracellular FREM2 Short and Smyth Nat Rev Nephr 12:754 2016 matrix protein 2 Glutamate receptor interacting (GRIP1) Short and Smyth Nat Rev Nephr 12:754 2016 protein 1 Short and Smyth Nat Rev Nephr 12:754 2016; ITGA8 Integrin α8 Schedl Nat Rev Genet 8:791 2007; Davis Pediatr Nephrol 29:597 2014 Woolf and Davies JASN 24:19 2013; Davis Pediatr (REN) Renin Nephrol 29:597 2014

Supplementary Table 3: Extracellular matrix components with a role in the development of the kidneys and urinary tract. Note that all murine and human phenotypes are recessive (autosomal or X-linked). Genes are divided into three groups: Fraser-complex related (light blue), Integrins/Laminins (light green), and HSPG-related (light orange) (see also Suppl. Fig. 3). Gene Protein Phenotype Mice Phenotype Human symbol Mutant mice exhibit significant Fraser Syndrome (OMIM amount of embryonic lethality due to Fraser 219000); isolated CAKUT hemorrhaging of embryonic blisters. extracellular (McGregor Nat Genet Survival is variable on genetic FRAS1 matrix 34:203 2003; van Haelst background. Further phenotypes complex Am J Med Genet A include severe renal developmental subunit 1 146A:2252 2008; Kohl defects and syndactyly. (McGregor JASN 25:1917 2014) Nat Genet 34:203 2003) Bifid nose with or without anorectal and renal anomalies (BNAR, OMIM Homozygous mice have 608980); Manitoba Fras1 related subepidermal blistering, oculotrichoanal syndrome extracellular FREM1 cryptophthalmos, syndactyly, and (MOTA, OMIM 248450); matrix renal agenesis. (Kiyozumi Proc Natl isolated CAKUT (Alazami protein 1 Acad Sci USA 103:11981 2006) Am J Hum Genet 85:414

2009; Slavotinek J Med Genet 48:375 2011; Kohl JASN 25:1917 2014) Homozygous mice display a significant amount of embryonic lethality due to hemorrhagic embryonic blisters. Renal Fraser Syndrome (OMIM

complex related complex Fras1 related - developmental defects and 219000); isolated CAKUT extracellular FREM2 syndactyly are common. (Jadeja Nat Genet 37:520 matrix Phenotypes of homozygous 2005; Kohl JASN 25:1917

Fraser protein 2 mutants are indistinguishable from 2014) those of Fras1 homozygous mutants. (Jadeja Nat Genet 37:520 2005) Fraser Syndrome (OMIM 219000); isolated CAKUT Glutamate Homozygous mice show increased (Vogel J Med Genet receptor embryonic lethality, blistering skin GRIP1 49:303 2012; Schanze Am interacting lesions and CAKUT. (Swiergiel Dev J Med Genet A 164A:837 protein 1 Dyn 219:21 2000) 2014; Kohl JASN 25:1917 2014) Homozygous mice die on the second day after birth. Survivors Isolated CAKUT (Humbert Integrin have reduced kidney size and ITGA8 Am J Hum Genet 94:288 alpha 8 abnormal steriocilia in the inner ear. 2014) (Muller Cell 88:6031997; Linton Development 134:2501 2007) Homozygous mice exhibit kidney agenesis or hypoplasia attributed to a delay in the invasion of the NPNT Nephronectin metanephric mesenchyme by the - ureteric bud at an early stage of kidney development. (Linton Development 134:2501 2007) Homozygous null mice die at or soon after implantation. Tissue- specific knockouts exhibit Integrin beta symptoms including skin blisters, ITGβ1 - 1 brain and heart defects, as well as

CAKUT phenotypes. (Wu Am J Physiol Renal Physiol 297:F210 2009) Homozygous null mice exhibit nothing reported in OMIM, symptoms including exencephaly, HGMD: Focal segmental Laminin syndactyly, CAKUT, and lethality in LAMA5 glomerulosclerosis alpha 5 late gestation. (Miner Dev Biol (Chatterjee Plos One 217:278 2000; Lo, MGI Ref ID 8:e76360 2013) Integrins, Laminins J:175213) Homozygous null mice display a nothing reported in OMIM, syndromic phenotype including Laminin HGMD: Dandy-Walker LAMC1 features from the CAKUT spectrum. gamma 1 malformation (Darbro Hum (Willem Development 129:2711 Mutat 34:1075 2013) 2002)

Supplementary Table 3 (contd.)

Gene Protein Phenotype Mice Phenotype Human symbol Homozygous mice display severe developmental defects Glucuronyl C5- including renal agenesis, lung Glce - epimerase abnormalities, and skeletal malformations. (Li J Biol Chem. 278:28363 2003) Simpson-Golabi- Behmel Syndrome, Type 1 (OMIM 312870) (Veugelers The gene trap mouse model Hum Mol Genet exibits neonatal lethality, 9:1321 2000; Baujat embryonic overgrowth and Am J Med Genet C Gpc3 Glypican 3 kidney cysts. The Gpc3 null 137C:4 2005; mouse exhibits enhanced UB Sakazume Am J Med branching. (Cano-Gauci J Cell Genet A 150B:151 Biol 146:255 1999) 2007; Kehrer Prenat Diagn 36:961 2016; Cotterau Am J Med Genet C 164A:282 2013) Homozygous mice exhibit symptoms including distended Urofacial Syndrome 1 urinary bladder, abnormal (OMIM 236730) (Pang voiding behavior, renal Hpse2 Heparanase 2 Am J Hum Genet dysfunction urinary bladder 86:957 2010; Stuart HSPG metabolism HSPG fibrosis, and lethality within one JASN 26:797 2015) month of age. (Guo Hum Mol Genet 24:1991 2015) Homozygous mice exhibit Heparan sulfate bilateral renal agenesis, bone 2-O- defects, eye development Hs2st1 - sulfotransferase abnormalities and cataracts. 1 (Bullock Genes Dev 15:1894 1998)

Homozygous mice die late in gestation or neonatally. They Mental retardation N- exhibit a mutisystemic (OMIM 616116) deacetylase/N- phenotype including (Reuter Am J Med Ndst1 sulfotransferase hydronephrosis and kidney Genet 164A:2753 (heparan cysts, respiratory distress and 2014; Najmabadi glucosaminyl) 1 failure. (Fan FEBS 467:7 2000; Nature 478:57 2011) Lo, MGI ID J:175213) Mice deficient in both genes exhibited highly penetrant neonatal lethality associated Sulfatase 1 + with multiple developmental Sulf1+Sulf2 - Sulfatase 2 defects including skeletal and renal abnormalities (CAKUT spectrum). (Holst Plos One 2:e575 2007)

Supplementary Table 4: Proteins that are related to BMP signaling and play a role in the development of the kidneys and the urinary tract. Corresponding genes/proteins are subcategorized in three groups: Regulators of BMP signaling (light red), BMP ligands (light blue), and BMP receptor(s) (light orange).

Reference Phenotype Human Gene Phenotype Protein Mice (contains CAKUT) symbol Mice MGI ID OMIM# Ikeya BMPER Development BMP-binding (Cross- Renal 133:4463 Diaphanospondylodysostosis endothelial veinless hypoplasia 2006; #608022 regulator 2) MGI 1920480 Failure of Sakaguchi postnatal CTD Nuclear Nat Commun CTDNEP1 nephron Envelope 4:1398 2013; - (Dullard) maintenance, Phosphatase MGI renal 1914431 hypoplasia. Chiu

Genesis Perinatal 50:711 2012, Cysteine rich lethality, Wilkinson transmembrane syndactyly, CRIM1 Kidney - BMP regulator and eye and Internat 1 kidney 76:1161 abnormalities. 2009; MGI 1354756 Renal Matzuk dysgenesis Nature (this FST Follistatin 374:360 - phenotype Regulators of BMP signalling BMP of Regulators 1995; MGI not reported 95586 in MGI) Cano-Gauci Renal J Cell Biol Simpson-Golabi-Behmel medullary GPC3 Glypican 3 146:255 syndrome, type 1, cystic 1999; MGI # 312870 dysplasia 104903 Michos Bilateral Development agenesis of 131:3401 isolated human CAKUT GREM1 Gremlin1 kidneys and 2004; (Kohl JASN 25:1917 2014) ureter MGI 1344337

Supplementary Table 4 (contd.)

Reference Phenotype Human Gene Gene name Phenotype Mice Mice (contains CAKUT) symbol MGI ID OMIM# Zhang and Bradley Homozygotes lethal Development between E7 and E10.5; 122:2977 Heterozygotes: Bone 1996; Hartwig Brachydactyly, increased proliferation BMP2 Morphogenic Mech Dev type A2; and branching of Protein 2 122:928 2005; #112600 ureteric bud Singh (this phenotpe not Sex Dev reported in MGI) 2:134 2008; MGI 88177 isolated human CAKUT Homozygotes lethal (Weber JASN

between E6.5 and E9.5; Miyazaki Bone 19:891 2008); Heterozygotes: renal J Clin Invest BMP4 Morphogenic syndromic abnormalities from 105:863 2000; Protein 4 microphthalmia 6 within the CAKUT MGI 88180 #607932; spectrum Orofacial cleft 1,

BMP ligands BMP #600625 Kingsley Cell Bone 71:399 1992; BMP5 Morphogenic Hydronephrosis King - Protein 5 Dev Biol 166:112 1994; MGI 88181 Dudley Genes Dev Bone Renal dysgenesis and 9:2795 1995; BMP7 Morphogenic hydroureter, arrested Luo - Protein 7 development Genes Dev 9:2808 1995; MGI 103302 Medullary hypoplasia and cortical cysts in

conditional knockout Hartwig J Am mutants in ureteric Soc Nephrol Bone Morpho- Polyposis epithelium; Renal 19:117 2008; genetic syndrome aplasia/dysgenesis or Hu BMPR1A Protein #174900; medullary dysplasia in Development Receptor #610069; mutants overexpressing 130:2753 Type 1A #174900 Bmpr1a throughout 2003; BMP receptor(s) BMP ureteric epithelium MGI 1338938 (this phenotype not reported in MGI)