Genetics of congenital anomalies of the kidney and urinary tract
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- Song, R. & Yosypiv, I.V. Pediatr Nephrol (2011) 26: 353. doi:10.1007/s00467-010-1629-4
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Congenital anomalies of the kidney and urinary tract (CAKUT) occur in 1 in 500 births and are a major cause of morbidity in children. Notably, CAKUT account for the most cases of pediatric end-stage renal disease and predispose the individual to hypertension and cardiovascular disease throughout life. Although some forms of CAKUT are a part of a syndrome or are associated with a positive family history, most cases of renal system anomalies are sporadic and isolated to the urinary tract. Broad phenotypic spectrum of CAKUT and variability in genotype–phenotype correlation indicate that pathogenesis of CAKUT is a complex process that depends on interplay of many factors. This review focuses on the genetic mechanisms (single-gene mutations, modifier genes) leading to renal system anomalies in humans and discusses emerging insights into the role of epigenetics, in utero environmental factors, and micro-RNAs (miRNAs) in the pathogenesis of CAKUT. Common gene networks that function in defined temporospatial fashion to orchestrate renal system morphogenesis are highlighted. Derangements in cellular, molecular, and morphogenetic mechanisms that direct normal renal system development are emphasized as a major cause of CAKUT. Integrated understanding of how morphogenetic process disruptions are linked to CAKUT will enable improved diagnosis, treatment, and prevention of congenital renal system anomalies and their consequences.
KeywordsKidney development CAKUT Renal hypodysplasia Renin-angiotensin
Nonsyndromic human congenital anomalies of the kidney and urinary tract (CAKUT)
Type of malformation
Anatomical and histological characteristics
No interaction between the UB and MM
Absence of the ureter and kidney
Aberrant interactions among the UB, MM or stroma
Reduced number of UB branches and nephrons that are fully formed, small kidney size
Aberrant interactions among the UB, MM or stroma
Reduced number of UB branches and nephrons. Presence of undifferentiated stromal and mesenchymal cells, cysts or cartilage. Frequently associated with kidney hypoplasia
Aberrant tubular and collecting duct patterning
Cysts in tubules and collecting ducts Normally formed glomeruli
Multicystic dysplastic kidneys
Aberrant interactions among the UB, MM or stroma
Absence of glomeruli and tubules
Presence of large cysts
Poorly formed atretic ureters
Small remnant kidney (if organ involutes)
Medullary cystic kidney disease 2
Aberrant tubular and collecting-duct patterning
Tubular atrophy, interstitial fibrosis, cysts in distal tubules and medullary collecting ducts
Supernumerary UB budding from the ND
Duplex ureters and kidneys or duplex ureters and collecting systems
May be associated with VUR or obstruction if UB budding is ectopic
Defects in renal capsule
Kidneys are fused at inferior lobes and located lower than usual
Morphologic development of the kidney and urinary tract
Molecular biology of mammalian kidney and urinary tract development
Genetic manipulations in mice provided critical information on the cellular and molecular mechanisms that orchestrate kidney and urinary tract morphogenesis, yielding new insights into the pathogenesis of CAKUT [6, 7, 8, 14]. Organogenesis of the metanephric kidney and lower urinary tract is coordinated by complex interactions among numerous transcription/growth factors and intracellular signaling molecules that may be collectively referred to as renal developmental genes (RDGs) [6, 7, 8, 14]. RDGs are expressed in the MM, stroma, angioblasts, UB, and cloaca [6, 7, 8, 9, 14]. Here, we review the most significant gene networks that function at multiple stages of renal system development.
UB induction from the ND is mediated by glial-derived neurotrophic factor (GDNF) secreted by MM, which interacts with the c-Ret receptor tyrosine kinase (RTK) expressed in the UB to induce branching [15, 16] (Fig. 1). GDNF levels and its spatial expression are regulated by multiple transcription and growth factors, such as paired box 2 (Pax2), eyes absent homolog 1 (Eya1), SIX homeobox (Six)1, 2, and 4, forkhead/winged-helix transcription factor d1 (FoxC1), roundabout, axon guidance receptor, homolog 2 (Robo2), slit homolog 2 (Slit2), wingless-type MMTV integration site family, member 11, (Wnt11) [6, 7]. c-Ret expression and signaling activity is inhibited by sprouty homolog 1 (Spry1) and induced by Pax2 or retinoic acid [17, 18, 19]. Interestingly, other growth factors, such as fibroblast growth factor (FGF) 10, can induce UB branching in the absence of GDNF or c-Ret RTK . Stromal cells stimulate UB branching and nephrogenesis via (Foxd1), retinoic acid, FGF7, bone morphogenetic protein 4 (BMP4), and podocyte 1 (Pod1) [21, 22, 23]. UB signals to MM by secreting Wnt9b, a soluble growth factor, that acts via canonical β-catenin to induce expression of FGF8, LIM homeobox 1 (Lhx1), and Wnt4 in the MM [24, 25, 26]. In turn, Wnt4 induces MM cells to undergo mesenchymal-to-epithelial transformation (MET) and differentiate into nephron epithelia . Wnt9b-induced MET is regulated by hepatocyte nuclear factor 1β (HNF1β) . In addition to its role in MET, Wnt9b is also required for later nephron patterning . Six2, a homeodomain transcription factor expressed in the MM, maintains MM cells in an undifferentiated state, thereby allowing continued UB branching and nephron formation to proceed . Iterative UB branching and nephron induction are accompanied by their continuous patterning (gradual acquisition of structure characteristic of the mature organ). Examples of patterning include nephron segmentation and UB remodeling into the renal collecting system. With respect to nephrogenesis, pretubular-mesenchymal-cell aggregates are induced at the tips of the UBs and then undergo MET to form the renal vesicle (RV) (Fig. 1). The RV elongates along the proximal–distal axis to form comma-shaped and then mature nephron, which comprises the following distinct regions: glomerulus, proximal tubule, loop of Henle, and distal tubule. Transition form the RV to comma-shaped nephron is induced by transcription factors Lhx1 and POU class 3 homeobox 3 (Brn1) . In turn, comma-shaped nephron elongates to form an S-shaped body. Distal parts of the S-shaped body differentiate into distal tubule segments under control of Brn1 and Iroquois-class homeodomain (Irx) transcription factors Irx1–3 , whereas proximal tubule formation is controlled by Notch2 . Notch2 controls proximal nephron development by stabilizing, rather than specifying, its fate via depletion of Six-2-positive progenitor cells . Podocyte differentiation is regulated by transcription factors Wilms tumor 1 (WT1), Pax2, LIM homeobox transcription factor 1β (Lmx1β), and semaphorin3a (Sema3a) [33, 34, 35]. Podocytes induce migration of vascular endothelial cells into the vascular cleft by releasing vascular endothelial growth factor (VEGF) . In turn, endothelial cells promote differentiation of mesangial cells via production of platelet-derived growth factor (PDGF) . Dissociation of the ureter from the ND and its fusion with the bladder is regulated by retinoic acid-, c-Ret-, and leukocyte antigen-related (LAR)-family protein tyrosine phosphatase-dependent apoptosis of the common ND [38, 39, 40]. Despite new insights into the molecular basis of kidney and urinary tract development, integrated understanding of this process is far from complete. Continued use of model organisms complemented by new technologies will enable identification of novel genes and factors that act in concert to form a kidney.
Developmental origin of CAKUT
Aberrant interactions between the UB and MM or stroma result in renal hypodysplasia, which is observed in 0.027% of fetuses by ultrasonography  (Fig. 2). Importantly, renal hypodysplasia due to decreased nephron endowment is causally linked to hypertension and eventual progression to chronic renal failure [48, 49]. Horseshoe kidney, which occurs in 1 in 1,000 newborns, may be caused by defects in renal capsule and failed migration due to aberrant stromal Foxd1 signaling [50, 51]. Nephron patterning defects may be due to aberrant cell proliferation, apoptosis, cell movements, or other causes. In this regard, altered balance of cell proliferation and apoptosis is responsible for abnormal patterning of the medulla in Beckwith–Wiedemann syndrome due to mutation in p57KIP2, an inhibitor of cell proliferation . Polycystic kidney disease (PKD), in which cysts arise from tubules that were formed normally in the context of development, represents another example of patterning defect. Here, increased tubular cell proliferation together with decreased integration of cells into the plane of tubular epithelium or loss of oriented cell division may account for postnatal cyst formation . PUV, observed in 0.003% of fetal ultrasounds, are due to aberrant cloacal development . A concerted effort to provide new insights into pathologic processes underlying the development of CAKUT is in progress within the Genitourinary Development Molecular Atlas Project (GUDMAP) .
Genetic mutations associated with human renal hypodysplasia
It is well appreciated that many instances of human CAKUT have a genetic basis and are associated with hereditary syndromes (Supplemental Table 1) . The inheritance pattern of some nonsyndromic cases of CAKUT (e.g. autosomal-dominant or -recessive polycystic kidney disease) is well known . Evidence is accumulating to suggest that mutations in select RDGs may be causally linked to the pathogenesis of nonsyndromic forms of CAKUT in humans (Table 1). Interestingly, mutations in a given RDG may be associated with a spectrum of urinary system anomalies. For example, Branchio-oto-renal (BOR) syndrome due to Eya1 mutations may be accompanied by renal agenesis or dysplasia, hypoplasia, or renal collecting system anomalies . It is therefore conceivable that Eya1 plays an important role at multiple stages of renal system morphogenesis. One of the mechanisms accounting for stage-specific impact of Eya1 mutation on renal system phenotype may involve cell-type-dependent interplay of up- or downstream regulators of Eya1. This possibility is supported by the findings that HNF1β, developmentally regulated transcription factor induces expression of uromodulin (UMOD) . Although HNF1β or UMOD mutations in humans cause renal hypodysplasia, the spectrum of disease phenotypes and age of their manifestations differ. HNF1β mutations manifest earlier in childhood and are associated with glomerulocystic kidney disease, unilateral multicystic dysplastic kidney (MCDK), uni- or bilateral cystic kidneys, renal hypodysplasia without cysts, horseshoe kidney, and oligomeganephronia [59, 60, 61, 62]. HNF1β mutations are detected in 33% of children with nonsyndromic MCDK or renal hypodysplasia . Most genetic alterations (64%) in this study were due to complete deletion of the gene, followed by truncating and nonsense mutations. No correlation between type of mutation and phenotype was observed. Interestingly, in 53% of probands, HNF1β mutations occurred de novo . These findings suggest a weak probability of genetic inheritance.
UMOD mutations are identified in medullary cystic kidney disease 2 (MCKD2) and familial juvenile hyperuricemic nephropathy, diseases that manifest later in life . In fact, no known UMOD mutations were identified in children with diverse forms of nonsyndromic CAKUT in a study by Wolf et al. . The authors proposed that unknown UMOD mutations may be causally linked to CAKUT in children. This possibility is supported by recent identification of a novel UMOD sequence variant in a child with decreased kidney function and hyperuricemia accompanied by histologic evidence of immature renal structures . In contrast to the impact of HNF1β or UMOD mutations on renal disease phenotype, Pax2, a transcription factor present in the metanephric mesenchyme, but not GDNF, polymorphism is associated with reduced kidney size in neonates [66, 67]. GDNF/c-Ret pathway is critical for metanephric development and GDNF expression is activated by Pax2 . Again, differential effects of Pax2 and GDNF polymorphism on renal phenotype may be due, in part, to unidentified cell-specific cofactors that regulate gene expression. Interestingly, RDG mutations associated with syndromal CAKUT [HNF1β, Eya1, Six1, sal-like 1 (Sall1), Pax2] are identified only in 5–15% of children from the European population with nonsyndromic CAKUT . Another recent study that examined children from the European population found no association of primary VUR with mutations in Pax2, HNF1β, c-Ret or Robo2 .
Several studies report a discrepancy in the impact of genetic mutations on renal-system phenotype between mice and humans. For example, despite severe renal phenotype observed in ROBO2/SLIT2-mutant mice, which includes formation of supernumerary ureters , these gene mutations are very rarely associated with familial nonsyndromic VUR in children [71, 72]. Similarly, mutations in homeobox A11 and D11(HoxA11/HoxD11), which cause renal hypoplasia in mice , are not associated with CAKUT in children . In addition, uroplakin (UP) UPII or UPIIIA mutations, which, respectively, cause hydronephrosis or VUR in mice , are not detected in the majority of children with nonsyndromic cases of CAKUT. However, UPII or UPIIIA mutations may account for CAKUT (e.g. MCDK or VUR) in a minority of these children [76, 77, 78]. Functionally, UPs account for impermeability of bladder epithelia to water. Mutations in Six2 and BMP4, which are expressed in the fetal kidney on weeks 9 and 12 of gestation, are identified in children with a broad spectrum of CAKUT ranging from unilateral or bilateral hypodysplasia to cystic dysplasia with or without VUR . Moreover, detected BMP4 mutations are associated with decreased BMP4 messenger RNA (mRNA) levels and enhanced protein degradation . Collectively, these data indicate that aberrant expression of RDGs is associated with diverse forms of CAKUT in humans. Interestingly, RDG mutations are detected in a minority of these patients . These observations point to the potential role for other mechanisms in the developmental origin of CAKUT.
Insights into the mechanisms controlling CAKUT phenotype
Renin–angiotensin system (RAS) gene mutations in human congenital anomalies of the kidney and urinary tract (CAKUT)
Renal system phenotype
Renal tubular dysgenesis: reduced number of proximal tubules, short proximal tubules without brush border, atrophic loops of Henle and collecting ducts, closely packed glomeruli, marked thickening and disorganization of interlobular and preglomerular arteries
Renal tubular dysgenesis
Renal tubular dysgenesis
Renal hypodysplasia due to posterior urethral valves
Renal tubular dysgenesis
UPJ stenosis, megaureter, MCDK, hydronephrosis, PUV
In polygenic diseases, mutations in multiple genes act in concert with environmental effects to cause a phenotype. With respect to renal system anomalies, intrauterine environment and fetal programming have been linked to CAKUT. Maternal low-protein diet initiated at onset of pregnancy reduces nephron number and alters gene expression in the embryonic metanephros in mice . Recent data indicate that environmental factors may act in concert with single-gene mutations to cause CAKUT. In this regard, renal hypodysplasia is caused by salt loading during embryonic development in bradykinin B2 receptor-deficient, but not in wild-type, mice . Exposure to cocaine or alcohol during gestation results in CAKUT in children [92, 93]. Importantly, the mechanistic basis for CAKUT associated with altered intrauterine environment remains to be elucidated.
There is a growing appreciation of the role of epigenetics in the regulation of gene expression and disease causality. The major players in epigenetic mechanisms of gene regulation are DNA or chromatin protein methylation, acetylation, and chromatin remodeling. Posttranslational modifications of histones such as histone acetylation and methylation may affect chromatin function and alter RDG expression in the absence of changes in DNA sequence [94, 95]. Relevant to pathogenesis of CAKUT, a recent study demonstrated a link between Pax2, a transcription factor critical for renal morphogenesis, and chromatin methylation . Gene expression may also be regulated at the posttranscriptional level by noncoding micro-RNAs (miRNAs). A critical role for miRNAs in kidney development is evident from the observation that targeted genetic inactivation of Dicer, the miRNA-processing enzyme, in UB in mice results in hydronephrosis . Collectively, these observations indicate that pathogenesis of CAKUT is a complex process that depends on the interplay of many factors. Moreover, despite advances in elucidation of the cellular and molecular mechanisms linking kidney organogenesis to CAKUT, our understanding of its cause in an individual patient is still too limited.
Renin–angiotensin system and CAKUT
The renin–angiotensin system (RAS) plays a major role in controlling blood pressure, fluid, and electrolyte homeostasis. The developing mammalian metanephros expresses all components of the RAS . Mutations in the genes encoding components of the RAS in mice cause diverse forms of CAKUT, which include hydronephrosis, hypoplastic medulla and papilla, marked thickening of renal arterial walls, duplex ureters, and vesicoureteral reflux [9, 97, 98]. Genetic manipulations in mice provided essential information regarding the mechanisms by which an intact RAS controls proper renal system development. The mechanistic basis of CAKUT due to RAS mutations may involve hypoplastic ureteral smooth-muscle layer, which exhibits impaired peristalsis , inhibition of Pax2 , GDNF/c-Ret [101, 102], or epidermal growth factor (EGF)-receptor  signaling, induction of BMP4 , decreased UB cell proliferation/survival resulting in reduced UB branching [101, 104] and reduced glomerular size . Reduction in EGF and PDGF expression in the medulla may account for renal papillary hypoplasia observed in angiotensinogen (AGT)-null mice . In addition, increased PDGF expression in endothelial cells may be responsible for thickening of the renal arteries observed in these mice .
With respect to epigenetic modifications of the RAS genes and its impact on CAKUT, recent studies in mice demonstrated that genetic inactivation of histone acetyltransferases such as cyclic adenosine monophosphate (cAMP)-response element-binding protein [CREB; (CBP)] and p300 alters kidney structure and results in a decreased number of renin-positive cells . Notably, kidneys of CBP/p300-null mice exhibit interstitial fibrosis, disorganized vasculature, and medullary and cortical cysts . In addition, targeted genetic inactivation of Dicer in juxtaglomerular cells causes a decrease in renin expression accompanied by striped fibrosis, a decrease in the number of renal vessels, and glomerular sclerosis . These data demonstrate that epigenetic modifications of the RAS and miRNAs play an important role in maintaining renal structural integrity.
Impact of RAS gene mutations on CAKUT in humans
All components of the RAS are expressed in the embryonic kidney as early as the fifth week of gestation [107, 108] when metanephric organogenesis is initiated . Initial evidence to indicate the critical role of the RAS in renal system organogenesis in humans was provided by the findings that the use of angiotensin-converting enzyme (ACE) inhibitors or AT1-receptor antagonists causes fetal anuria, leading to oligohydramnios [109, 110]. Evidence is emerging to suggest that mutations in the RAS genes may be causally linked to the pathogenesis of CAKUT in humans. In this regard, homozygous or compound heterozygous mutations in the genes encoding for AGT, renin, ACE, or AT1 receptors in French children are associated with renal tubular dysgenesis (RTD) . Detected DNA mutations include the following nucleotide alterations: 1124G>A (AGT), 145C>T, 689G>A, 310G>A (renin), 798C>G (ACE), 845C>T (AT1R). In RTD, the kidneys are usually of normal size. Renal cortex exhibits a paucity of proximal tubules. In the medulla, collapsed collecting ducts and abundant interstitial fibrosis are observed . Vascular changes involve thickening of arterial walls. Glomerular number may be normal, low, or increased, whereas glomerular size is normal or enlarged. In addition, all affected individuals exhibit hypotension. Importantly, the majority of children with RTD die in the perinatal period due to anuria combined with pulmonary hypoplasia. Few patients survive beyond the neonatal period. However, no correlation has been identified so far between the type of mutation and clinical course.
Association of single polymorphism in the human AT2R gene, 1332A>G transition, with CAKUT have been reported in several, but not all, studies. This alteration in the AT2R is described in American and German Caucasian males with ureteropelvic junction (UPJ) stenosis, megaureter, and MCDK [98, 113]. The same transition was detected in Korean children with unilateral MCDK, renal agenesis, and hydronephrosis ; in Italian children with VUR, hypoplastic kidney, UPJ stenosis, nonobstructive megaureter, and PUV , and in Serbian children with UPJ, VUR, or megaureter . In contrast, there was no association of primary VUR with mutations in AT2R in a recent European study . This difference may be due to an insufficient number of patients studied, limitations in study design, low penetrance of AT2R mutation, contribution of modifier genes, or other factors. ACE polymorphism is associated with kidney hypodysplasia caused by posterior urethral valves . Collectively, emerging evidence points to an important role for the intact RAS in structural maturation of the kidney and urinary tract in humans.
The reasons for different renal phenotypes, particularly in papillary hypoplasia and hydronephrosis observed in mice but not in humans with RAS gene mutations are not clear. Additional discrepancy includes the presence of severe proximal tubular damage in humans and apparent absence of these changes in mice. One potential mechanism to explain these differences may include timing in completion of nephrogenesis. In this regard, new nephron formation ceases at 36 weeks of gestation in humans but continues for 2 weeks after birth in mice. It is conceivable that postnatal rise in renal blood flow (RBF) contributes to normal proximal tubule patterning in mice. In addition, rising RBF may enhance the postnatal urine flow rate, which, together with decreased ureteral peristalsis , will cause hydronephrosis.
Given the broad spectrum of human CAKUT and variable clinical impact of different forms of renal system anomalies ranging from mild asymptomatic cases to severe kidney injury manifesting before or after birth, each patient with CAKUT requires individualized clinical management. Because morbidity in mild cases of CAKUT may not manifest until later in life, these patients should be closely followed throughout life. Medical monitoring should include diet, nutritional status, growth, blood pressure, renal function, proteinuria, and urinary tract imaging. New biomarkers are needed to better assess disease progression and therapeutic strategies (e.g. prenatal correction of obstruction or use of renin system inhibitors to slow progression of renal injury to preserve renal function). Genetic counseling is recommended for all patients with familial cases of CAKUT or newly diagnosed forms of CAKUT that suggest presence of genetic anomalies. Introduction of more sensitive array-based methods that allow screening for multiple gene mutations and unravel a complex network of molecular interactions will help determine and predict occurrence and consequences of CAKUT. Finally, establishment of shared large biorepositories of patients with CAKUT for molecular, genetic, and translational studies will have a major impact on designing novel strategies to prevent and manage CAKUT.