Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) are the commonest cause of chronic kidney disease in children. Structural anomalies within the CAKUT spectrum include renal agenesis, kidney hypo-/dysplasia, multicystic kidney dysplasia, duplex collecting system, posterior urethral valves and ureter abnormalities. While most CAKUT cases are sporadic, familial clustering of CAKUT is common, emphasizing a strong genetic contribution to CAKUT origin. Animal experiments demonstrate that alterations in genes crucial for kidney development can cause experimental CAKUT, while expression studies implicate mislocalization and/or aberrant levels of the encoded proteins in human CAKUT. Further insight into the pathogenesis of CAKUT will improve strategies for early diagnosis, follow-up and treatment. Here, we outline a collaborative approach to identify and characterize novel factors underlying human CAKUT. This European consortium will share the largest collection of CAKUT patients available worldwide and undertake multidisciplinary research into molecular and genetic pathogenesis, with extension into translational studies to improve long-term patient outcomes.

Introduction

Congenital anomalies of the kidney and urinary tract

Congenital anomalies of the kidney and urinary tract (CAKUT) comprise a broad spectrum of renal and urinary tract malformations that altogether occur in ∼1:500 live-born fetuses and cause neonatal death in 1:2000 births [ 1 ]. Structural anomalies of the CAKUT spectrum encompass renal agenesis, renal hypo-/dysplasia, multicystic kidney dysplasia, duplex renal collecting system, ureteropelvic junction obstruction, mega-ureter, posterior urethral valves (PUV) and vesicoureteral reflux (VUR). Renal abnormalities are found in close relatives in ∼10% of CAKUT patients [ 2 ], although these are frequently asymptomatic. Hence, inherited cases may be missed and the frequency of familial CAKUT under-estimated. Although CAKUT typically occur as isolated malformations, they occasionally develop in association with additional congenital abnormalities outside the urinary tract, for example, in the renal coloboma syndrome and the renal cysts and diabetes syndrome [ 3–6 ]. CAKUT commonly cause progressive chronic kidney disease (CKD) and constitute the most frequent cause of end-stage renal failure (CKD Stage 5) and renal replacement therapy in childhood [ 7 , 8 ]. Understanding of the causes of CAKUT is essential for adequate disease stratification and prediction of prognosis [ 9 ]. Recently, several European research groups have agreed to join forces and resources to establish a large CAKUT patient registry and a co-ordinated research programme. Here, this European CAKUT (EUCAKUT) consortium discusses novel perspectives for the identification and functional characterization of important factors involved in nephrogenesis and CAKUT pathogenesis. We outline how better understanding of the molecular pathways involved in kidney development contributes to the design of new diagnostic tools and will explore opportunities for the development of innovative therapeutic approaches.

Impact of CAKUT on quality of life and mortality

CAKUT is the principal cause of end-stage renal failure [ 7 , 10 , 11 ]. The prevalence of children with severe forms of CAKUT tends to increase due to improved peri- and post-natal management. Due to their lifelong exposure to CKD and its sequelae, individuals affected by CAKUT suffer from excessive morbidity and mortality. Furthermore, CKD and the need for kidney replacement therapy in childhood lead to severe impairment of physical development and psychosocial integration [ 12 ]. Patients suffer from growth retardation, impaired sexual maturation and numerous other partially disabling multisystem complications, many of which continue into adult life and compromise their chances of leading a successful work and social life. From a societal perspective, CAKUT pose a significant economic burden on health care systems related to the patients’ lifelong costly therapeutic needs and severely impact the employment potential of affected individuals and their families. Moreover, childhood-onset CKD is associated with accelerated cardiovascular disease, resulting in a serious risk of cardiovascular events at young adult age [ 13 ]. These manifold consequences of CAKUT provide a firm rationale for experimental and clinical research exploring novel diagnostic, preventive and therapeutic avenues in order to improve the outcomes of this disorder [ 14 ].

The EUCAKUT consortium

The EUCAKUT consortium is a complementary multidisciplinary collaboration of clinical researchers, geneticists, developmental biologists and bioinformaticians. The consortium aims to identify complex molecular networks that underlie CAKUT pathogenesis, the natural course of CAKUT and the mechanisms responsible for individual phenotypic characteristics ( Figure 1 ). The consortium puts effort in recruiting a unique study population, currently comprising well over 2000 well-defined CAKUT patients, encompassing the complete spectrum of disease.

Fig. 1.

Novel perspectives investigating CAKUT.

Fig. 1.

Novel perspectives investigating CAKUT.

Development of the kidney and urinary tract in health and disease

Normal nephrogenesis

Development of the mammalian kidney and urinary tract requires complex interactions and signalling processes between several embryonic tissues [ 15–17 ]. The kidneys and upper urinary tract arise from the Wolffian duct and the metanephric mesenchyme, both originating from the intermediate mesoderm. Major events during kidney development are the formation of the Wolffian duct, the intense communication between ureteric bud and metanephric mesenchyme cells and distal ureter maturation [ 18 ]. After the extension of the Wolffian duct caudally along the body axis, ureteric bud formation is initiated. The ureteric bud grows into the metanephric blastema, stimulating a proportion of mesenchyme cells to undergo mesenchyme epithelial transformation and differentiation into specialized nephron segments [ 19 , 20 ]. At the same time, the cells of the metanephric mesenchyme induce ureteric bud growth and branching, involving cell proliferation and differentiation, giving rise to the epithelium of the renal pelvis, the ureter, the bladder trigone and the renal collecting ducts. Ureteric bud branching is related to the eventual nephron number. Kidney induction and differentiation involve multiple gene networks including transcription factors, cell adhesion molecules, growth factors, cell-polarity molecules, components of the Wnt signalling pathway and renin–angiotensin system (RAS) and additional inducing factors [ 4 , 15 ]. Many genes involved in nephrogenesis have been identified, but we are far from a complete understanding of their inter-relationships within complex networks and the temporal controls underlying embryonic kidney and urinary tract formation [ 18 , 21 ].

Disrupted nephrogenesis

Structural renal anomalies can arise from defects in nephrogenesis [ 15 ]. Genetic as well as environmental factors that are present before or during pregnancy are presumed to be involved in deficient kidney development [ 22 , 23 ], as multiple key molecules essential in urinary tract morphogenesis have been identified from animal models displaying urinary tract malformations [ 15 ]. It is assumed that the majority of renal agenesis is caused by the absence of Wolffian duct induction or by lack of interactions between the metanephric mesenchyme and the ureteric bud [ 18 ]. Excessive ureteric bud branching might result in duplex collecting system, whereas VUR is likely to have its origin in the stage of kidney and ureter maturation [ 18 ]. It has been debated whether PUV is part of the CAKUT phenotype spectrum as well. This specific phenotype might have a different underlying cause. However, co-occurrence of PUV and other uropathies belonging to the CAKUT spectrum plead for similar backgrounds.

Identification of key factors involved in CAKUT pathogenesis

Complex origin of CAKUT

The occurrence of syndrome phenotypes and familial clustering proves that there are genetic causes for CAKUT [ 24 ]. However, the CAKUT spectrum encompasses disease traits that often do not follow classical Mendelian inheritance patterns. The complex character of CAKUT is underlined by the variable CAKUT phenotypes among family members with the same single gene defect, ranging from asymptomatic structural abnormalities to severe renal insufficiency [ 23 , 25 ]. Thus, it is hypothesized that CAKUT aetiology matches a range of monogenic to complex inheritance patterns, involving genetic and environmental underlying factors [ 26 ]. So far, linkage analysis, association studies and candidate gene approaches have been applied in the search for novel CAKUT risk factors, resulting in the identification of several genes implicated in CAKUT pathogenesis ( Tables 1 and 2 ) [ 35 , 46 , 49 ]. In most cases, however, gene defects could not be identified, underlining the major heterogeneity and complex mechanisms underlying CAKUT. Here, we summarize results from linkage analyses, association studies, candidate gene approaches and investigations of environmental predisposing factors.

Table 1.

Genetic loci a for congenital anomalies of the kidney and urinary tract, identified by (non) parametric linkage analyses

Locus CAKUT phenotype Model Parametric (H)LOD score NPL (P-value) Reference 
1p13 VUR Autosomal dominant 3.16 5.76 (0.0002)  [ 27 ]  
1p32–33 Renal agenesis, renal hypoplasia Autosomal dominant 3.50 5.30 (0.00015)  [ 28 ]  
1q41–44 and 11p11 PUV and Prune Belly syndrome Autosomal recessive 3.01 –  [ 29 ]  
2q37 VUR Non-parametric – 4.10 (0.001)  [ 30 ]  
6p21 Hydronephrosis, UPJ obstruction Autosomal dominant 3.09 –  [ 31 ]  
8q24 Renal agenesis, VUR Autosomal recessive 4.20 –  [ 32 ]  
12p11–q13 VUR Autosomal recessive 3.60 4.00 (0.0001)  [ 33 ]  
Locus CAKUT phenotype Model Parametric (H)LOD score NPL (P-value) Reference 
1p13 VUR Autosomal dominant 3.16 5.76 (0.0002)  [ 27 ]  
1p32–33 Renal agenesis, renal hypoplasia Autosomal dominant 3.50 5.30 (0.00015)  [ 28 ]  
1q41–44 and 11p11 PUV and Prune Belly syndrome Autosomal recessive 3.01 –  [ 29 ]  
2q37 VUR Non-parametric – 4.10 (0.001)  [ 30 ]  
6p21 Hydronephrosis, UPJ obstruction Autosomal dominant 3.09 –  [ 31 ]  
8q24 Renal agenesis, VUR Autosomal recessive 4.20 –  [ 32 ]  
12p11–q13 VUR Autosomal recessive 3.60 4.00 (0.0001)  [ 33 ]  
a

Significant/suggestive association was favoured over completeness in reporting interesting genetic loci for CAKUT. NPL, non-parametric linkage; (H)LOD, (heterogeneity) logarithm of the odds.

Table 2.

Genes investigated in unrelated patients with isolated congenital anomalies of the kidney and urinary tract

Gene CAKUT phenotype  Type of mutations identified a Mutation detection rate in unrelated cases Reference 
BMP4 Renal hypoplasia Missense 5/250 (2%)  [ 34 ]  
EYA1 Renal hypoplasia Insertion, deletion 2/99 (2%)  [ 35 ]  
GDNF Renal agenesis, renal dysplasia Missense 1/33 (3%)  [ 36 ]  
GFRA1 Renal agenesis, renal dysplasia None 0/33 (0%)  [ 36 ]  
HNF1β Renal agenesis, renal hypoplasia, renal dysplasia Deletion, splice site 8/99 (8%), 75/377 (20%), 5/50 (10%), 25/80 (31%)  [ 35 , 37–39 ]  
HOXA11/HOXD11 Renal agenesis, renal hypoplasia, renal dysplasia None 0/59 (0%)  [ 40 ]  
PAX2 Renal hypoplasia, renal dysplasia Insertion, deletion, splice site, stop 6/99 (6%), 2/20 (10%), respectively  [ 35 , 41 ]  
RET Renal agenesis, renal dysplasia Missense, stop 9/33 (27%), 7/101 (7%)  [ 36 , 42 ]  
ROBO2 VUR Missense 6/95 (6%)  [ 43 ]  
SALL1 Renal hypoplasia Deletion 1/99 (1%)  [ 35 ]  
SIX1 Renal hypoplasia Missense 1/99 (1%)  [ 35 ]  
SIX2 Renal hypoplasia Missense 5/250 (2%)  [ 34 ]  
SOX17 VUR, UPJ obstruction Missense, insertion 6/178 (3%)  [ 44 ]  
UMOD Complete CAKUT spectrum None 0/96 (0%)  [ 45 ]  
UPK3A Renal agenesis, renal dysplasia, renal hypoplasia, PUV, VUR Missense 0/76 (0%), 2/170 (1%), 4/17 (24%)  [ 46 , 47 , 48 ]  
Gene CAKUT phenotype  Type of mutations identified a Mutation detection rate in unrelated cases Reference 
BMP4 Renal hypoplasia Missense 5/250 (2%)  [ 34 ]  
EYA1 Renal hypoplasia Insertion, deletion 2/99 (2%)  [ 35 ]  
GDNF Renal agenesis, renal dysplasia Missense 1/33 (3%)  [ 36 ]  
GFRA1 Renal agenesis, renal dysplasia None 0/33 (0%)  [ 36 ]  
HNF1β Renal agenesis, renal hypoplasia, renal dysplasia Deletion, splice site 8/99 (8%), 75/377 (20%), 5/50 (10%), 25/80 (31%)  [ 35 , 37–39 ]  
HOXA11/HOXD11 Renal agenesis, renal hypoplasia, renal dysplasia None 0/59 (0%)  [ 40 ]  
PAX2 Renal hypoplasia, renal dysplasia Insertion, deletion, splice site, stop 6/99 (6%), 2/20 (10%), respectively  [ 35 , 41 ]  
RET Renal agenesis, renal dysplasia Missense, stop 9/33 (27%), 7/101 (7%)  [ 36 , 42 ]  
ROBO2 VUR Missense 6/95 (6%)  [ 43 ]  
SALL1 Renal hypoplasia Deletion 1/99 (1%)  [ 35 ]  
SIX1 Renal hypoplasia Missense 1/99 (1%)  [ 35 ]  
SIX2 Renal hypoplasia Missense 5/250 (2%)  [ 34 ]  
SOX17 VUR, UPJ obstruction Missense, insertion 6/178 (3%)  [ 44 ]  
UMOD Complete CAKUT spectrum None 0/96 (0%)  [ 45 ]  
UPK3A Renal agenesis, renal dysplasia, renal hypoplasia, PUV, VUR Missense 0/76 (0%), 2/170 (1%), 4/17 (24%)  [ 46 , 47 , 48 ]  
a

All mutations were found in heterozygous states, fitting a dominant disease model.

Linkage and association analyses in CAKUT gene identification

Previous efforts to ascertain genetic causes by linkage analysis in families and association studies in large case–control settings identified causal variants in only a very small proportion of CAKUT patients [ 34 , 35 , 49–51 ]. Reported evidence for genetic loci associated with non-syndromal, familial CAKUT is displayed in Table 1 , but no disease-causing genes have been identified from these loci thus far. Additionally, array-based comparative genomic hybridization in 30 children with syndromal CAKUT phenotypes with at least one additional extra-renal symptom reported DNA micro-imbalances in three patients (10%), identifying novel chromosomal regions associated with CAKUT [ 50 ]. In 2010, the UK VUR Study Group reported a combined linkage and family-based association approach in 320 families with primary non-syndromic VUR [ 49 ]. They concluded that major genetic loci may not exist for VUR within European populations, although linkage was demonstrated for multiple genetic loci and several single nucleotide polymorphisms (SNPs) were associated. Genetic heterogeneity was further emphasized by a linkage study investigating 12 candidate genes and two reported loci in four large VUR families, excluding GDNF , RET , SLIT2 , SPRY1 , PAX2 , AGTR2 , UPK1A , UPK3A , 1p13 and 20p13 from linkage to VUR [ 52 ].

Candidate gene approach in CAKUT patient cohorts

Candidate genes of interest for sequence analysis in large well-characterized CAKUT cohorts include genes: (i) identified in family studies, (ii) important during nephrogenesis, (iii) causing syndromal forms of CAKUT, (iv) known to cause CAKUT when mutated in animal models and (v) known to be deregulated in human CAKUT from expression studies. The European multicentre ESCAPE study screened five renal developmental genes ( HNF1β , PAX2 , EYA1 , SIX1 and SALL1 ) in 100 children with renal hypodysplasia [ 35 ]. Novel variants were detected in 17% of the patients, with most (15%) variants identified in HNF1β and PAX2 . The HNF1β gene encodes a transcription factor expressed in the epithelial cells of the kidney, pancreas, liver and lung, which is essential for the formation of tubular structures in these organs in very early embryogenesis [ 53 ]. Mutations in human HNF1β cause the renal cysts and diabetes syndrome (MIM#137920), an autosomal-dominant disorder that presents with abnormal kidney development and/or diabetes, which may occur at young age [ 3 ]. HNF1β mutations range from single point mutations to partial or complete gene deletions. PAX2 is another transcription factor, expressed in the developing kidney, eye and ear and is crucial for cell proliferation and induction of nephrogenesis [ 54 , 55 ]. Gene defects in human PAX2 are associated with the renal coloboma syndrome (MIM#120330), including renal hypoplasia. Interestingly, HNF1β and PAX2 mutations were also detected in non-syndromic CAKUT patients, but no genotype–phenotype correlations were found [ 37 , 38 , 41 , 56 ]. Current DNA diagnostics for renal tract abnormalities mainly focuses on HNF1β and PAX2 screening. However, the majority of CAKUT cases remain unexplained. Candidate genes that were previously investigated in CAKUT patient cohorts are outlined in Table 2 . Taken together, these data suggest that many candidate genes are implicated in CAKUT, which underlines the heterogeneous background of CAKUT.

Next generation sequencing: a novel strategy in the search for CAKUT genes

Revolutionary sequencing techniques became accessible very recently, bringing along great opportunities for novel disease gene identification. In particular, the powerful combination of next generation sequencing and genome-wide studies such as linkage analysis in multiplex families is promising for the revelation of gene defects and molecular networks involved in CAKUT aetiology. Next generation sequencing strategies involving targeted genomic enrichment enable deep sequencing of explicit parts of the genome [ 57 , 58 ], for example, the exome that comprises all the exons (∼180 000) of the genome. Recent studies emphasized that exome capture and sequencing in selected patient-groups and families has great potential in the discovery of causal genes in complex traits as well as monogenic conditions [ 59 , 60 ]. We hypothesize that major risk allele identification in multiplex families also indicates the involvement of common variants in the same genes in sporadic cases. Thus, high-throughput mutation screening in multiple CAKUT candidate genes by targeted sequencing, followed by functional characterization studies will reveal the importance of inclusion of particular genes in DNA diagnostics. For the benefit of CAKUT patients and their relatives, we see great opportunities for the development of a massively parallel sequencing strategy in CAKUT diagnostics which enables effective concurrent screening of all known CAKUT disease genes in multiple individual DNA samples. Translation of next generation sequencing outcomes also enables development of genetic tools for risk profile determination, indicating disease progression and outcome. Future implementation in clinical practice might then involve a personalized medicine approach with individual follow-up strategies, fine tuning of drug and surgical intervention, gene therapy and renal support based on the genetic profile of the affected individual or family. Results from CAKUT studies involving next generation sequencing will pave the way towards further characterization studies on the identified genes, pathways and biological systems that are crucial for normal renal function.

Environmental risk factors for CAKUT

An important role is expected for environmental factors in CAKUT aetiology, based on the incomplete penetrance of disease in CAKUT families and the occurrence of diverse phenotypes within pedigrees [ 23 , 25 ]. Thus, it is hypothesized that specific mutations can potentially affect the development of the urinary tract, with the final phenotypic outcome depending on modifying factors. Associations were previously found between CAKUT and intrauterine conditions like maternal diabetes [ 61 ], the diet of the mother [ 62 ] and maternal drug use with agents affecting the RAS [ 63 , 64 ]. However, strong evidence is still scarce. Crucial is the large-scale collection of uniform questionnaire data on occupational and environmental factors present before and during pregnancy from both parents in large cohorts of CAKUT patients and families. Such an approach already facilitated experimental gene–environment interaction studies to reveal novel aetiologic pathways [ 22 ]. Through the collection of exhaustive questionnaire data on environmental and occupational factors across the large CAKUT series, an incredibly valuable data set is being created, which will be available for studying the wide variety of potential environmental factors involved. Moreover, different factors in separate regions across Europe or between various ethnic backgrounds may be identified.

Functional characterization of novel CAKUT key factors

Characterization of the genes involved in kidney development

For further characterization of the genes involved in normal and disrupted mammalian kidney development, gene expression profiling in mouse embryonic kidneys and functional gene characterization in organotypic embryonic kidney mouse models are the most promising tools. Determination of the nephrogenesis-related gene function is managed by both ‘loss-of-function’ and ‘gain-of-function’ experiments performed on embryonic kidneys of wild-type and transgenic mice. A recently developed method, involving dissociation and reintegration of mouse embryonic kidney cells in the presence of growth factors that evoke competence for nephrogenesis, enables efficient viral transduction of the cells [ 65 ]. We see further implications for in vivo morpholino-mediated knockdown approaches in mouse embryonic kidney cultures, providing ample opportunities for semi- and high-throughput screening of the most important factors in nephrogenesis [ 66 ]. It is important to develop novel tools aimed at the detection of normal and impaired gene activity and classifying the impact on kidney formation. In this perspective, detailed mouse embryonic kidney culture development can be studied with high-resolution light and epifluorescent microscopy in real-time and time-lapse mode. This provides spatial–time characterization of nephrogenic induction and branching morphogenesis [ 67 ]. The Wnt signalling pathway, and in particular the Wnt-4 gene, is a key element in regulating metanephric induction and nephrogenesis in general [ 67–69 ]. Better understanding of Wnt signalling events in the developing kidney and especially the identification of target genes is in focus of the EUCAKUT research consortium.

The role of transcription factors in nephrogenesis

Organ growth requires the precise co-ordination of gene expression, which is largely governed by transcription factors. As such, it is not surprising that a significant number of mutations responsible for developmental renal defects are found in genes coding for transcriptional regulators [ 15 ]. While in the past transcriptional dependencies have been determined using candidate target gene approaches, it is now clear that transcriptional regulation is much more complex involving highly sophisticated networks with intricate interdependencies. To fully comprehend these interactions in kidney development, the most appropriate modern technology to identify target genes on a genome-wide level is high-throughput techniques such as in vivo chromatin immunoprecipitation (ChIP) Seq analysis [ 70 ]. This technique provides a direct reading of active regulatory processes as well as the position of important enhancers within a locus by the isolation and identification of sequences bound by transcription factors within a specific cell type. The challenge for the future is integration of the enormous data sets obtained from multiple ChIP-Seq experiments into comprehensive models by expert bioinformaticians. Novel algorithms need to be developed that allow the discovery of regulatory networks and the prediction of transcription factor interactions in multi-protein complexes which regulate targets via cell type-specific enhancer elements during nephrogenesis.

In vivo CAKUT disease modelling

While in vitro analysis contributes insight into the molecular roles of individual genes, in vivo characterization is required to fully understand the role of a gene in normal development and its interaction with other genetic factors. Classically, functional analysis is performed in mice based on similar urogenital tract architecture compared to man and the relative ease by which the mouse genome can be modified via gene targeting. Since the principal organization of the nephron has been strongly conserved during evolution in other vertebrate systems as well, such as zebrafish ( Danio rero ) and frog ( Xenopus ), they represent powerful alternatives, in particular when analysing the role of genes involved in nephron patterning. Data sets obtained by various approaches are useful to address specific questions, but it is only by combining them that their full potential can be harvested. Ultimately, it will be essential to integrate the data obtained by molecular, genomic and functional approaches into a single database. An intuitive way forward would be the mapping of data to a histological database such as the 3D EuReGene kidney atlas ( http://www.euregene.org/portal/pages/index.html ). This would allow query of any given structure or cell type regarding gene expression, transcriptional regulation, functional data and associated diseases.

Gene expression analysis in normal human fetal renal tissues and kidney organ culture

Animal experiments allow precise targeting and manipulation of key factors involved in kidney development, but these studies need corroboration in human CAKUT. Several unique human resources are currently available, including normal human fetal tissues between 6 and 14 weeks gestation and later-stage (ab)normal fetal and post-natal kidney samples that facilitate extensive investigations on human CAKUT gene expression, localization and quantification. Furthermore, a recently generated unique collection of normal and dysplastic human fetal progenitor cell lines facilitates further functional characterization studies [ 71 , 72 ]. Prospective collection of amniotic fluid (AF) stem cells is ongoing, with the aim to bioengineer patients’ own cells for future personal treatment. In this perspective, new inducers of differentiation are being tested in vitro . Additionally, mouse human chimaeras are being developed, using both a re-aggregation assay in vitro [ 65 ] and injection under the capsule of newborn mice in vivo . Human-induced pluripotent stem (iPS) cells derived from CAKUT patients provide another tremendous potential for models that phenocopy aspects of developmental defects. Altogether, these models will help to further investigate findings from CAKUT gene discoveries and animal studies, with the final aim to translate study results to the human situation.

Implications for iPS cell technology in CAKUT therapy

Following a better understanding of the genetic and cellular basis of CAKUT, new cell therapeutic methods are under development. A similar strategy is applied in both in vitro and in vivo models of CAKUT: specific targeted cell populations are ablated in mice, after which the impact on kidney development and function is evaluated. Next, the ablated cells are replaced by corresponding wild-type cells, aiming to rescue the disrupted developmental programme or nephropathy. In this approach, it is essential to select the correct types and generations of cells that are most suitable for cell replacement and transplantation purposes. Currently, mouse embryonic kidney cells and renal mesenchymal stem cells are being used. However, it is likely that iPS cells will also provide a good source for transplantation. For this purpose, in vitro differentiation of human iPS cells into different renal lineage cells that are suitable for transplantation will be established and functionally tested in vitro and in vivo . Interestingly, cell replacement technologies can be applied in vivo to generate animals with ‘humanized’ organs. This technology has already demonstrated its high potential in generation of mice with humanized liver [ 73 ] and pancreas [ 74 ]. Innovative cell replacement methodologies aim at kidney tissue and organ engineering, opening new avenues for generation of complete kidneys suitable for transplantation [ 75 , 76 ].

Prenatal imaging of the developing kidney and CAKUT

Routine prenatal ultrasound examination and advanced ultrasound equipment result in a prenatal detection rate of ∼89% of renal and urinary tract malformations [ 77 ]. Accurate diagnosis is important for counselling of the parents and planning of multidisciplinary care for the newborn. Antenatal investigations that predict post-natal renal function present varying accuracy of outcomes mainly based on the amount of AF and the morphological appearance of the renal system in association with fetal serum and urine markers. This requires invasive testing and the clinical usefulness to predict fetal renal function is currently insufficiently proven [ 78 , 79 ]. To improve the prediction of fetal renal function, 3D-techniques investigate fetal urinary production by calculating bladder volumes and evaluate the prognostic value of the residual renal cortex volume. Renal perfusion is measured by pulsed Doppler analysis of both renal vessels and by colour Doppler analysis of the renal cortex of each kidney in association with renal cortex volume. This approach could enhance the prediction of individual renal function. Fetal magnetic resonance (MR) spectroscopy and functional MR may develop into prognostic non-invasive tools to guide prenatal treatment in well-defined cases. For implementation in cases of lethal renal pathology, the diagnostic value of virtual MRI autopsy in addition to targeted organ biopsy is currently under investigation as an alternative for conventional autopsy.

Prognostic biomarkers for CAKUT

Hippocrates linked health problems to diagnostic changes in the urine already two millennia ago and now measurement of urinary components such as protein, using dipsticks and standard laboratory tests, is routine in diagnostics. However, these tests are quite crude; since there has to be significant kidney damage before frank proteinuria occurs. Therefore, identification and implementation of novel early prognostic markers would enable more accurate investigation, for example using rapidly advancing proteomics techniques in (i) urine from children with CAKUT where urine collection is routine and less invasive than blood tests and (ii) AF for antenatally diagnosed CAKUT cases where AF is often aspirated for genetic analysis A pilot study in 10 CAKUT patients and six control AF samples using 2D DiGE and shotgun proteomics identified six molecules that were deregulated in CAKUT patients emphasize progress in the development of new biomarkers (Heywood, Mills, Wang, Winyard and UK Renal Association, unpublished work). Evolving technology will allow analysis of up to 30 different marker molecules in individual samples within minutes, which is very promising for the development and application of a reproducible assay, screening important prognostic factors for CAKUT in routine clinical practice.

Future perspectives in CAKUT research

Understanding the complex mechanisms involved in normal kidney development and pathogenesis of CAKUT will contribute to improvement of health-related quality of life for CAKUT patients. In this perspective, we see great opportunities for the use of a massively parallel sequencing screening method in prenatal and post-natal CAKUT diagnostics. Deep sequencing of multiple CAKUT-predisposing genes in a single person’s DNA and measurements on genetic and biochemical biomarkers may contribute to early diagnosis, culminating in personalized medicine involving not just genetic counselling for individual CAKUT patients and their family members [ 14 ] but also bespoke therapy for their condition. Of crucial importance for the understanding of mechanisms underlying disease progression towards CKD Stage 5 and thus, insight into prognostic factors, requires accurate longitudinal follow-up of CAKUT patients. Current nephroprotective treatment involves surgical intervention, management of hypertension and episodes of urinary tract infection, inhibition of the RAS and administration of supplements for renal support. To decide whether therapeutic strategies can be adjusted based on individual genetic profiles in the future, further research is needed. In this context, the EUCAKUT consortium forms an excellent setting for the implementation of joint efforts in daily clinical practice.

Dutch Kidney Foundation—KSTP10.004 (K.Y.R., N.V.A.M.K. and E.M.H.F.B.). Nephro-Urology Unit, UCL Institute of Child Health, London (P.J.W.). Academy of Finland, European Union (Star-T-Rek), Finnish Cancer Foundation and Sigrid Juselius Foundation (I.N.S. and S.V.). Foundation of the Scientific Research Flandres (E.L.). Clinical Research Fund of The Catholic University Leuven (A.H.). Fritz-Thyssen-Stiftung (S.W. and F.S.). Fondation du Rein, Agence Nationale de la Recherche and Fondation de la Recherche Médicale (A.S.). Institut National de la Santé et de la Recherche Médicale, Agence Nationale de la Recherche - ANR06-MRAR-034-01, ANR07-MRAR-010-01, GIS-Institut des Maladies Rares—AAE07007KSA and Programme Hospitalier de la Recherche Clinique Assistance Publique—AOM07129 (C.J., R.S. and C.A.). Dietmar Hopp-Stiftung (Nephrogen), KfH-Stiftung für Präventivmedizin (4C Study) and ERA-EDTA Research Programme (4C Study) (F.S.). Academy of Finland Centre of Excellence Programme 2012-2017 (S.V.).

Conflict of interest statement . None declared.

References

1.
Loane
M
Dolk
H
Kelly
A
, et al.  . 
Paper 4: EUROCAT statistical monitoring: identification and investigation of ten year trends of congenital anomalies in Europe
Birth Defects Res A Clin Mol Teratol
 , 
2011
, vol. 
91
 
Suppl 1
(pg. 
S31
-
S43
)
2.
Winyard
P
Chitty
LS
Dysplastic kidneys
Semin Fetal Neonatal Med
 , 
2008
, vol. 
13
 (pg. 
142
-
151
)
3.
Horikawa
Y
Iwasaki
N
Hara
M
, et al.  . 
Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY
Nat Genet
 , 
1997
, vol. 
17
 (pg. 
384
-
385
)
4.
Sanna-Cherchi
S
Caridi
G
Weng
PL
, et al.  . 
Genetic approaches to human renal agenesis/hypoplasia and dysplasia
Pediatr Nephrol
 , 
2007
, vol. 
22
 (pg. 
1675
-
1684
)
5.
Woolf
AS
Price
KL
Scambler
PJ
, et al.  . 
Evolving concepts in human renal dysplasia
J Am Soc Nephrol
 , 
2004
, vol. 
15
 (pg. 
998
-
1007
)
6.
Sanyanusin
P
Schimmenti
LA
McNoe
LA
, et al.  . 
Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux
Nat Genet
 , 
1995
, vol. 
9
 (pg. 
358
-
364
)
7.
Sanna-Cherchi
S
Ravani
P
Corbani
V
, et al.  . 
Renal outcome in patients with congenital anomalies of the kidney and urinary tract
Kidney Int
 , 
2009
, vol. 
76
 (pg. 
528
-
533
)
8.
Mansoor
O
Chandar
J
Rodriguez
MM
, et al.  . 
Long-term risk of chronic kidney disease in unilateral multicystic dysplastic kidney
Pediatr Nephrol
 , 
2011
, vol. 
26
 (pg. 
597
-
603
)
9.
Hildebrandt
F
Genetic kidney diseases
Lancet
 , 
2010
, vol. 
375
 (pg. 
1287
-
1295
)
10.
Kerecuk
L
Schreuder
MF
Woolf
AS
Renal tract malformations: perspectives for nephrologists
Nat Clin Pract Nephrol
 , 
2008
, vol. 
4
 (pg. 
312
-
325
)
11.
Hains
DS
Bates
CM
Ingraham
S
, et al.  . 
Management and etiology of the unilateral multicystic dysplastic kidney: a review
Pediatr Nephrol
 , 
2009
, vol. 
24
 (pg. 
233
-
241
)
12.
Groothoff
JW
Long-term outcomes of children with end-stage renal disease
Pediatr Nephrol
 , 
2005
, vol. 
20
 (pg. 
849
-
853
)
13.
Krediet
RT
Balafa
O
Cardiovascular risk in the peritoneal dialysis patient
Nat Rev Nephrol
 , 
2010
, vol. 
6
 (pg. 
451
-
460
)
14.
James
MT
Hemmelgarn
BR
Tonelli
M
Early recognition and prevention of chronic kidney disease
Lancet
 , 
2010
, vol. 
375
 (pg. 
1296
-
1309
)
15.
Schedl
A
Renal abnormalities and their developmental origin
Nat Rev Genet
 , 
2007
, vol. 
8
 (pg. 
791
-
802
)
16.
Woolf
AS
A molecular and genetic view of human renal and urinary tract malformations
Kidney Int
 , 
2000
, vol. 
58
 (pg. 
500
-
512
)
17.
Vainio
S
Lin
Y
Coordinating early kidney development: lessons from gene targeting
Nat Rev Genet
 , 
2002
, vol. 
3
 (pg. 
533
-
543
)
18.
Uetani
N
Bouchard
M
Plumbing in the embryo: developmental defects of the urinary tracts
Clin Genet
 , 
2009
, vol. 
75
 (pg. 
307
-
317
)
19.
Schmidt-Ott
KM
Yang
J
Chen
X
, et al.  . 
Novel regulators of kidney development from the tips of the ureteric bud
J Am Soc Nephrol
 , 
2005
, vol. 
16
 (pg. 
1993
-
2002
)
20.
Hu
MC
Rosenblum
ND
Genetic regulation of branching morphogenesis: lessons learned from loss-of-function phenotypes
Pediatr Res
 , 
2003
, vol. 
54
 (pg. 
433
-
438
)
21.
Rumballe
B
Georgas
K
Wilkinson
L
, et al.  . 
Molecular anatomy of the kidney: what have we learned from gene expression and functional genomics?
Pediatr Nephrol
 , 
2010
, vol. 
25
 (pg. 
1005
-
1016
)
22.
El-Dahr
SS
Harrison-Bernard
LM
Dipp
S
, et al.  . 
Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development
Physiol Genomics
 , 
2000
, vol. 
3
 (pg. 
121
-
131
)
23.
Schwaderer
AL
Bates
CM
McHugh
KM
, et al.  . 
Renal anomalies in family members of infants with bilateral renal agenesis/adysplasia
Pediatr Nephrol
 , 
2007
, vol. 
22
 (pg. 
52
-
56
)
24.
Belk
RA
Thomas
DF
Mueller
RF
, et al.  . 
A family study and the natural history of prenatally detected unilateral multicystic dysplastic kidney
J Urol
 , 
2002
, vol. 
167
 (pg. 
666
-
669
)
25.
Kerecuk
L
Sajoo
A
McGregor
L
, et al.  . 
Autosomal dominant inheritance of non-syndromic renal hypoplasia and dysplasia: dramatic variation in clinical severity in a single kindred
Nephrol Dial Transplant
 , 
2007
, vol. 
22
 (pg. 
259
-
263
)
26.
Ichikawa
I
Kuwayama
F
Pope
JC
, et al.  . 
Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT
Kidney Int
 , 
2002
, vol. 
61
 (pg. 
889
-
898
)
27.
Feather
SA
Malcolm
S
Woolf
AS
, et al.  . 
Primary, nonsyndromic vesicoureteric reflux and its nephropathy is genetically heterogeneous, with a locus on chromosome 1
Am J Hum Genet
 , 
2000
, vol. 
66
 (pg. 
1420
-
1425
)
28.
Sanna-Cherchi
S
Caridi
G
Weng
PL
, et al.  . 
Localization of a gene for nonsyndromic renal hypodysplasia to chromosome 1p32-33
Am J Hum Genet
 , 
2007
, vol. 
80
 (pg. 
539
-
549
)
29.
Weber
S
Mir
S
Schlingmann
KP
, et al.  . 
Gene locus ambiguity in posterior urethral valves/prune-belly syndrome
Pediatr Nephrol
 , 
2005
, vol. 
20
 (pg. 
1036
-
1042
)
30.
Kelly
H
Molony
CM
Darlow
JM
, et al.  . 
A genome-wide scan for genes involved in primary vesicoureteric reflux
J Med Genet
 , 
2007
, vol. 
44
 (pg. 
710
-
717
)
31.
Izquierdo
L
Porteous
M
Paramo
PG
, et al.  . 
Evidence for genetic heterogeneity in hereditary hydronephrosis caused by pelvi-ureteric junction obstruction, with one locus assigned to chromosome 6p
Hum Genet
 , 
1992
, vol. 
89
 (pg. 
557
-
560
)
32.
Ashraf
S
Hoskins
BE
Chaib
H
, et al.  . 
Mapping of a new locus for congenital anomalies of the kidney and urinary tract on chromosome 8q24
Nephrol Dial Transplant
 , 
2010
, vol. 
25
 (pg. 
1496
-
1501
)
33.
Weng
PL
Sanna-Cherchi
S
Hensle
T
, et al.  . 
A recessive gene for primary vesicoureteral reflux maps to chromosome 12p11-q13
J Am Soc Nephrol
 , 
2009
, vol. 
20
 (pg. 
1633
-
1640
)
34.
Weber
S
Taylor
JC
Winyard
P
, et al.  . 
SIX2 and BMP4 mutations associate with anomalous kidney development
J Am Soc Nephrol
 , 
2008
, vol. 
19
 (pg. 
891
-
903
)
35.
Weber
S
Moriniere
V
Knuppel
T
, et al.  . 
Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study
J Am Soc Nephrol
 , 
2006
, vol. 
17
 (pg. 
2864
-
2870
)
36.
Skinner
MA
Safford
SD
Reeves
JG
, et al.  . 
Renal aplasia in humans is associated with RET mutations
Am J Hum Genet
 , 
2008
, vol. 
82
 (pg. 
344
-
351
)
37.
Heidet
L
Decramer
S
Pawtowski
A
, et al.  . 
Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases
Clin J Am Soc Nephrol
 , 
2010
, vol. 
5
 (pg. 
1079
-
1090
)
38.
Nakayama
M
Nozu
K
Goto
Y
, et al.  . 
HNF1B alterations associated with congenital anomalies of the kidney and urinary tract
Pediatr Nephrol
 , 
2010
, vol. 
25
 (pg. 
1073
-
1079
)
39.
Ulinski
T
Lescure
S
Beaufils
S
, et al.  . 
Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort
J Am Soc Nephrol
 , 
2006
, vol. 
17
 (pg. 
497
-
503
)
40.
Bouba
I
Siomou
E
Stefanidis
CJ
, et al.  . 
Absence of mutations in the HOXA11 and HOXD11 genes in children with congenital renal malformations
Pediatr Nephrol
 , 
2009
, vol. 
24
 (pg. 
1569
-
1572
)
41.
Negrisolo
S
Benetti
E
Centi
S
, et al.  . 
PAX2 gene mutations in pediatric and young adult transplant recipients: kidney and urinary tract malformations without ocular anomalies
Clin Genet
 , 
2010
, vol. 
80
 (pg. 
581
-
585
)
42.
Jeanpierre
C
Mace
G
Parisot
M
, et al.  . 
RET and GDNF mutations are rare in fetuses with renal agenesis or other severe kidney development defects
J Med Genet
 , 
2011
, vol. 
48
 (pg. 
497
-
504
)
43.
Bertoli-Avella
AM
Conte
ML
Punzo
F
, et al.  . 
ROBO2 gene variants are associated with familial vesicoureteral reflux
J Am Soc Nephrol
 , 
2008
, vol. 
19
 (pg. 
825
-
831
)
44.
Gimelli
S
Caridi
G
Beri
S
, et al.  . 
Mutations in SOX17 are associated with congenital anomalies of the kidney and the urinary tract
Hum Mutat
 , 
2010
, vol. 
31
 (pg. 
1352
-
1359
)
45.
Wolf
MT
Hoskins
BE
Beck
BB
, et al.  . 
Mutation analysis of the Uromodulin gene in 96 individuals with urinary tract anomalies (CAKUT)
Pediatr Nephrol
 , 
2009
, vol. 
24
 (pg. 
55
-
60
)
46.
Jiang
S
Gitlin
J
Deng
FM
, et al.  . 
Lack of major involvement of human uroplakin genes in vesicoureteral reflux: implications for disease heterogeneity
Kidney Int
 , 
2004
, vol. 
66
 (pg. 
10
-
19
)
47.
Schonfelder
EM
Knuppel
T
Tasic
V
, et al.  . 
Mutations in Uroplakin IIIA are a rare cause of renal hypodysplasia in humans
Am J Kidney Dis
 , 
2006
, vol. 
47
 (pg. 
1004
-
1012
)
48.
Jenkins
D
Bitner-Glindzicz
M
Malcolm
S
, et al.  . 
De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure
J Am Soc Nephrol
 , 
2005
, vol. 
16
 (pg. 
2141
-
2149
)
49.
Cordell
HJ
Darlay
R
Charoen
P
, et al.  . 
Whole-genome linkage and association scan in primary, nonsyndromic vesicoureteric reflux
J Am Soc Nephrol
 , 
2010
, vol. 
21
 (pg. 
113
-
123
)
50.
Weber
S
Landwehr
C
Renkert
M
, et al.  . 
Mapping candidate regions and genes for congenital anomalies of the kidneys and urinary tract (CAKUT) by array-based comparative genomic hybridization
Nephrol Dial Transplant
 , 
2011
, vol. 
26
 (pg. 
136
-
143
)
51.
Lu
W
van Eerde
AM
Fan
X
, et al.  . 
Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux
Am J Hum Genet
 , 
2007
, vol. 
80
 (pg. 
616
-
632
)
52.
van Eerde
AM
Koeleman
BP
van de Kamp
JM
, et al.  . 
Linkage study of 14 candidate genes and loci in four large Dutch families with vesico-ureteral reflux
Pediatr Nephrol
 , 
2007
, vol. 
22
 (pg. 
1129
-
1133
)
53.
Coffinier
C
Barra
J
Babinet
C
, et al.  . 
Expression of the vHNF1/HNF1beta homeoprotein gene during mouse organogenesis
Mech Dev
 , 
1999
, vol. 
89
 (pg. 
211
-
213
)
54.
Stapleton
P
Weith
A
Urbanek
P
, et al.  . 
Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9
Nat Genet
 , 
1993
, vol. 
3
 (pg. 
292
-
298
)
55.
Tellier
AL
Amiel
J
Delezoide
AL
, et al.  . 
Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome
Am J Med Genet
 , 
2000
, vol. 
93
 (pg. 
85
-
88
)
56.
Edghill
EL
Bingham
C
Ellard
S
, et al.  . 
Mutations in hepatocyte nuclear factor-1beta and their related phenotypes
J Med Genet
 , 
2006
, vol. 
43
 (pg. 
84
-
90
)
57.
Turner
EH
Ng
SB
Nickerson
DA
, et al.  . 
Methods for genomic partitioning
Annu Rev Genomics Hum Genet
 , 
2009
, vol. 
10
 (pg. 
263
-
284
)
58.
Hoischen
A
Gilissen
C
Arts
P
, et al.  . 
Massively parallel sequencing of ataxia genes after array-based enrichment
Hum Mutat
 , 
2010
, vol. 
31
 (pg. 
494
-
499
)
59.
Gilissen
C
Arts
HH
Hoischen
A
, et al.  . 
Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome
Am J Hum Genet
 , 
2010
, vol. 
87
 (pg. 
418
-
423
)
60.
Yi
X
Liang
Y
Huerta-Sanchez
E
, et al.  . 
Sequencing of 50 human exomes reveals adaptation to high altitude
Science
 , 
2010
, vol. 
329
 (pg. 
75
-
78
)
61.
Davis
EM
Peck
JD
Thompson
D
, et al.  . 
Maternal diabetes and renal agenesis/dysgenesis
Birth Defects Res A Clin Mol Teratol
 , 
2010
, vol. 
88
 (pg. 
722
-
727
)
62.
Welham
SJ
Riley
PR
Wade
A
, et al.  . 
Maternal diet programs embryonic kidney gene expression
Physiol Genomics
 , 
2005
, vol. 
22
 (pg. 
48
-
56
)
63.
Schreuder
MF
Bueters
RR
Huigen
MC
, et al.  . 
Effect of Drugs on Renal Development
Clin J Am Soc Nephrol
 , 
2010
, vol. 
6
 (pg. 
212
-
217
)
64.
Sekine
T
Miura
K
Takahashi
K
, et al.  . 
Children's toxicology from bench to bed–drug-induced renal injury (1): the toxic effects of ARB/ACEI on fetal kidney development
J Toxicol Sci
 , 
2009
, vol. 
34
 
Suppl 2
(pg. 
SP245
-
SP250
)
65.
Unbekandt
M
Davies
JA
Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues
Kidney Int
 , 
2010
, vol. 
77
 (pg. 
407
-
416
)
66.
Hartwig
S
Ho
J
Pandey
P
, et al.  . 
Genomic characterization of Wilms′ tumor suppressor 1 targets in nephron progenitor cells during kidney development
Development
 , 
2010
, vol. 
137
 (pg. 
1189
-
1203
)
67.
Shan
J
Jokela
T
Skovorodkin
I
, et al.  . 
Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development
Differentiation
 , 
2010
, vol. 
79
 (pg. 
57
-
64
)
68.
Stark
K
Vainio
S
Vassileva
G
, et al.  . 
Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4
Nature
 , 
1994
, vol. 
372
 (pg. 
679
-
683
)
69.
Itaranta
P
Chi
L
Seppanen
T
, et al.  . 
Wnt-4 signaling is involved in the control of smooth muscle cell fate via Bmp-4 in the medullary stroma of the developing kidney
Dev Biol
 , 
2006
, vol. 
293
 (pg. 
473
-
483
)
70.
Visel
A
Blow
MJ
Li
Z
, et al.  . 
ChIP-seq accurately predicts tissue-specific activity of enhancers
Nature
 , 
2009
, vol. 
457
 (pg. 
854
-
858
)
71.
Romio
L
Wright
V
Price
K
, et al.  . 
OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells
J Am Soc Nephrol
 , 
2003
, vol. 
14
 (pg. 
680
-
689
)
72.
Price
KL
Long
DA
Jina
N
, et al.  . 
Microarray interrogation of human metanephric mesenchymal cells highlights potentially important molecules in vivo
Physiol Genomics
 , 
2007
, vol. 
28
 (pg. 
193
-
202
)
73.
Strom
SC
Davila
J
Grompe
M
Chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity
Methods Mol Biol
 , 
2010
, vol. 
640
 (pg. 
491
-
509
)
74.
Stanger
BZ
Tanaka
AJ
Melton
DA
Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver
Nature
 , 
2007
, vol. 
445
 (pg. 
886
-
891
)
75.
Nakauchi
H
Kobayashi
T
Lee
Y
, et al.  . 
Organ regeneration method utilizing blastocyst complementation
 
76.
Nakauchi
H
Kobayashi
T
Yamaguchi
T
, et al.  . 
Organ regeneration method utilizing iPS cell and blastocyst complementation
 
77.
Levi
S
Mass screening for fetal malformations: the Eurofetus study
Ultrasound Obstet Gynecol
 , 
2003
, vol. 
22
 (pg. 
555
-
558
)
78.
Morris
RK
Malin
GL
Khan
KS
, et al.  . 
Systematic review of the effectiveness of antenatal intervention for the treatment of congenital lower urinary tract obstruction
BJOG
 , 
2010
, vol. 
117
 (pg. 
382
-
390
)
79.
Berry
SM
Lecolier
B
Smith
RS
, et al.  . 
Predictive value of fetal serum beta 2-microglobulin for neonatal renal function
Lancet
 , 
1995
, vol. 
345
 (pg. 
1277
-
1278
)

Comments

0 Comments