Impact of next generation sequencing on our understanding of CAKUT.

Congenital abnormalities of the kidney and urinary tract (CAKUT) form the leading cause of pediatric end-stage renal disease. Knowledge on the molecular mechanisms that underlie CAKUT leads to the improvement of DNA diagnostics and counseling regarding prognosis and recurrence risk estimation for CAKUT patients and their relatives. Implementation of next generation sequencing in research and diagnostic settings has led to the identification of the molecular basis of many developmental diseases. In this review, we summarize the efforts on next generation sequencing in CAKUT research and we discuss how next generation sequencing added to our understanding of CAKUT genetics. Although next generation sequencing has certainly proven to be a game changer in the field of disease gene identification and novel CAKUT-causing gene variants have been identified, most CAKUT cases still remain unsolved. Occurring with genetic and phenotypic heterogeneity along with incomplete penetrance, the identification of CAKUT etiology poses many challenges. We see great potential for combined -omics approaches that include next generation sequencing in the identification of CAKUT-specific biomarkers, which is necessary to optimize the care for CAKUT patients.

Interkinetic nuclear movements promote apical expansion in pseudostratified epithelia at the expense of apicobasal elongation.

Pseudostratified epithelia (PSE) are a common type of columnar epithelia found in a wealth of embryonic and adult tissues such as ectodermal placodes, the trachea, the ureter, the gut and the neuroepithelium. PSE are characterized by the choreographed displacement of cells' nuclei along the apicobasal axis according to phases of their cell cycle. Such movements, called interkinetic movements (INM), have been proposed to influence tissue expansion and shape and suggested as culprit in several congenital diseases such as CAKUT (Congenital anomalies of kidney and urinary tract) and esophageal atresia. INM rely on cytoskeleton dynamics just as adhesion, contractility and mitosis do. Therefore, long term impairment of INM without affecting proliferation and adhesion is currently technically unachievable. Here we bypassed this hurdle by generating a 2D agent-based model of a proliferating PSE and compared its output to the growth of the chick neuroepithelium to assess the interplay between INM and these other important cell processes during growth of a PSE. We found that INM directly generates apical expansion and apical nuclear crowding. In addition, our data strongly suggest that apicobasal elongation of cells is not an emerging property of a proliferative PSE but rather requires a specific elongation program. We then discuss how such program might functionally link INM, tissue growth and differentiation.

In utero myelomeningocoele repair and urological outcomes: the first 100 cases of a prospective analysis. Is there an improvement in bladder function?

OBJECTIVES: To evaluate the first 100 cases of in utero myelomeningocoele (MMC) repair and urological outcomes in a prospective analysis aiming to define possible improvement in bladder function. PATIENTS AND METHODS: We used a protocol consisting of a detailed medical history, urinary tract ultrasonography, voiding cystourethrography, and urodynamic evaluation. Patients were categorised into four groups: normal, high risk (overactive bladder with a detrusor leak-point pressure >40 cm H2 O and high filling pressures also >40 cm H2 O), incontinent, and underactivity (underactive bladder with post-void residual urine), and patients were treated accordingly. RESULTS: We evaluated 100 patients, at a mean age of 5.8 months (median 4 months), classified as high risk in 52.6%, incontinent in 27.4%, with underactive bladder in 4.2%, and only 14.7% had a normal bladder profile. Clean intermittent catheterisation was initiated in 57.3% of the patients and anticholinergics in 52.6%. Antibiotic prophylaxis was initiated in 19.1% of the patients presenting with vesico-ureteric reflux. CONCLUSION: The high incidence of abnormal bladder patterns suggests little benefit of in utero MMC surgery concerning the urinary tract.

Morphological study of the ureterovesical junction in children.

Morphological and morphometric assessment of the elements of the ureterovesical junction in children was performed in the present study in different age groups ranging from 24 weeks of gestation to 16 years old. We tried to answer the question whether, in human ontogenesis, there is a period of anatomical predisposition to primary vesicoureteral reflux. The study included 210 urinary bladders with juxtavesical parts of the ureters that had been obtained from routine autopsies. As a result of the study, we showed that provided the pregnancy is uncomplicated there is no inherited susceptibility that would account for reducing or disturbing the development of ureterovesical junction elements. Based on the analysis of our results concerning the anatomy and morphological changes taking place in the elements of the ureterovesical junction in different age groups, one could put forward a hypothesis that anatomical predisposition to primary vesicoureteral reflux occurs in age group II, i.e. between 28 and 37 weeks of gestation. The parameters that define interrelations between elements of the ureterovesical junction and that are considered essential for normal functioning of antireflux mechanism were still observed in age group VI in our study. This suggests that these relations are not unambiguous in children with normally structured and functionally competent vesicoureteral junction.

Heterozygous loss-of-function mutation in Odd-skipped related 1 (Osr1) is associated with vesicoureteric reflux, duplex systems, and hydronephrosis.

Odd-skipped related 1 (Osr1) is a transcriptional repressor that plays critical roles in maintaining the mesenchymal stem cell population within the developing kidney. Here, we report that newborn pups with a heterozygous null mutation in Osr1 exhibit a 21% incidence of vesicoureteric reflux and have hydronephrosis and urinary tract duplications. Newborn pups have a short intravesical ureter, resulting in a less competent ureterovesical junction which arises from a delay in urinary tract development. We describe a new domain of Osr1 expression in the ureteral mesenchyme and within the developing bladder in the mouse. OSR1 was sequenced in 186 children with primary vesicoureteric reflux, and 17 have single nucleotide polymorphisms. Fifteen children have a common synonymous variant, rs12329305, one child has a rare nonsynonymous variant, rs3440471, and one child has a rare 5'-UTR variant, rs45535040 The impact of these SNPs is not clear; therefore, the role of OSR1 in human disease remains to be elucidated. Osr1 is a candidate gene implicated in the pathogenesis of vesicoureteric reflux and congenital abnormalities of the kidney and urinary tract in mice.

Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.

Outcome and etiologies of fetal megacystis according to the gestational age at diagnosis.

OBJECTIVE: To investigate the gestational age-specific outcomes and the different etiologies of megacystis diagnosed at screening ultrasound. METHODS: A retrospective single-center study was conducted between 1989 and 2009. We identified all consecutive cases of megacystis prenatally diagnosed during routine ultrasound screening. Outcome, final diagnosis, and renal function were recorded. RESULTS: Eighty-four patients were included. An isolated lower urinary tract obstruction was observed in 38/84 (45.2%), ureterovesical reflux in 9/84 (10.7%), an associated congenital abnormality in 32/84 (38.1%) and a normal bladder in 5/84 (6%). Increased gestational age at diagnosis was correlated with an increased rate of live born children (P < 0.01). No cases of megacystis diagnosed in the first trimester were born alive. When diagnosis of posterior urethral valves (PUV) was made in the third trimester, the ultimate survival rate was 11/13 (84.6%) compared with 3/12 (25%) for a diagnosis made in the second trimester (P = 0.02). CONCLUSION: Lower urinary tract obstruction is the main etiology of megacystis. Megacystis can also be part of more complex malformations. Outcome of megacystis detected in the first trimester is poor. PUV detected in the third trimester had a better overall survival rate than PUV detected in the second trimester.

The C3H/HeJ inbred mouse is a model of vesico-ureteric reflux with a susceptibility locus on chromosome 12.

Vesico-ureteric reflux is the most common congenital anomaly of the urinary tract, characterized by a defective uretero-vesical junction with retrograde urine flow from the bladder toward the kidneys. Because there is strong evidence for a genetic basis for some cases of vesico-ureteric reflux, we screened 11 inbred mouse strains for reflux and kidney size and identified one strain, C3H/HeJ, that has a 100 percent incidence of vesico-ureteric reflux with otherwise normal kidneys at birth. These mice are predisposed to reflux as a result of a defective uretero-vesical junction characterized by a short intravesical ureter. This defect results from a delay in urinary tract development initially manifested by a ureteric bud arising from a more caudal location along the mesonephric duct. In contrast, C57BL/6J mice (resistant to reflux at birth) have long intravesical ureters, normally positioned ureteric buds, and no delay in urinary tract development. Genome-wide and additional fine mapping of backcross mice, derived from C3H/HeJ and C57BL/6J crosses, identified a significant reflux susceptibility locus, Vurm1, on chromosome 12 (peak logarithm of the odds=7.39). The C3H/HeJ mouse is a model of vesico-ureteric reflux without renal malformation, and further characterization of this model will allow for the identification of a pathway important for urinary tract development, a finding that will serve as a model for the human disorder.

Correlation of prenatal and postnatal ultrasound findings with the incidence of vesicoureteral reflux in children with fetal renal pelvic dilatation.

PURPOSE: Up to 1% of prenatal ultrasounds detect renal pelvic dilatation. This dilatation is associated with vesicoureteral reflux but its clinical significance and the necessity for vesicoureteral reflux detection have been questioned. We report an evaluation of fetal renal pelvic dilatation and postnatal sonographic features with the incidence of vesicoureteral reflux. MATERIALS AND METHODS: Maximum fetal renal pelvic dilatation was prospectively measured at a single center between 1990 and 2003. Dilatation 4 mm or greater at less than 33 weeks of gestation, or 7 mm or greater at more than 33 weeks was the threshold for inclusion in the study. Postnatal evaluation included ultrasound and voiding cystourethrogram. Postnatal data included vesicoureteral reflux incidence and grade, and caliceal and ureteral dilatation on ultrasound. RESULTS: Of 215 neonates 46 (21%) had vesicoureteral reflux. Mean renal pelvic dilatation was 14.4 mm in those with reflux, which was not statistically different than the mean of 11.8 mm in 169 with a normal voiding cystourethrogram. ROC analysis revealed that fetal renal pelvic dilatation was a poor discriminator of reflux. Reflux was identified in a significantly greater number of neonates with vs without postnatal calicectasis (20% vs 9%, p <0.05). When fetal renal pelvic dilatation was combined with postnatal calicectasis, only 5% of infants with dilatation less than 10 mm and isolated renal pelvic dilatation had reflux, whereas reflux was identified in 25% with fetal renal pelvic dilatation 10 mm or greater and calicectasis (p <0.02). CONCLUSIONS: The magnitude of fetal renal pelvic dilatation is not reliably predictive of reflux and this measure alone cannot be used to direct postnatal cystography. However, postnatal calicectasis appears to be an important predictor of vesicoureteral reflux in children with fetal renal pelvic dilatation. Expectant management can be considered in the subset of newborns with minimal dilatation (less than 10 mm) and absent calicectasis.

A genome-wide scan for genes involved in primary vesicoureteric reflux.

BACKGROUND: Vesicoureteric reflux (VUR) is the retrograde flow of urine from the bladder into the ureters. It is the most common urological anomaly in children, and a major cause of end-stage renal failure and hypertension in both children and adults. VUR is seen in approximately 1-2% of Caucasian newborns and is frequently familial. OBJECTIVE AND METHODS: In order to search for genetic loci involved in VUR, we performed a genome-wide linkage scan using 4710 single-nucleotide polymorphisms (SNPs) in 609 individuals from 129 Irish families with >1 affected member. RESULTS: Nonparametric linkage (NPL) analysis of the dataset yielded moderately suggestive linkage at chromosome 2q37 (NPL(max) = 2.67, p<0.001). Analysis of a subset without any additional features, such as duplex kidneys, yielded a maximum NPL score of 4.1 (p = 0.001), reaching levels of genome-wide statistical significance. Suggestive linkage was also seen at 10q26 and 6q27, and there were several smaller peaks. CONCLUSION: Our results confirm the previous conclusion that VUR is genetically heterogeneous, and support the identification of several disease-associated regions indicated by smaller studies, as well as indicating new regions of interest for investigation.

Embryologic basis for lower ureteral anomalies: a hypothesis.

A hypothesis for the embryogenesis of lower ureteral anomalies invokes variations in location and number of ureteral buds on the mesonephric duct. Such vagaries determine the length of the common nephric duct and the time of meeting of the ureteral bud (or buds) with the urogenital sinus. These factors in turn affect the density of the mesenchymal tissue and its mass in relation to the adjoining structures and, ultimately, the musculature of the trigone as well as the level of the ureteral hiatus and the musculature of the bladder base. Primary reflux, ureteral ectopy, ureteral duplication, ureterocele, functional ureteral obstruction, and congenital strictures are viewed and possibly explained in the light of the embryonal variations mentioned.

Abnormalities of the ureteral bud.

The development and incorporation of the ureteric bud into the developing bladder is a complex process, and it is influenced by events occurring at the same time in the development of other systems in the perineum. Knowledge of the embryology of the ureteric bud and associated wolffian duct is the key to understanding the development of the entire genitourinary system. If the normal embryology is understood, the dysembryogenesis that results in many of the common anomalies of the lower urinary system is simplified. Many of the concepts proposed in this discussion are well substantiated, others are more difficult to prove conclusively. It is believed, however, that the embryologic approach provides a simplified understanding of many of the common problems encountered in pediatric urology. The key in clinical practice to the diagnosis and understanding of these defects is the cystoscopic examination. Careful assessment of the orifice's position, appearance, and tunnel length will allow the interpretation of how the abnormality came about, and also provides valuable information concerning the state of the associated renal segment. This in turn allow better therapeutic management of the problem based on the knowledge of the severity of the renal anomaly associated with the abnormality found in the bladder.

Greater reliability of neonatal ultrasonography in defining renal hypoplasia with antenatal hydronephrosis and vesicoureteral reflux.

PURPOSE: Infants with history of antenatal hydronephrosis and neonatal vesicoureteral reflux may have detectable changes in renal scans before the advent of urinary tract infection. In cases of bilateral high-grade vesicoureteral reflux, differential renal function on renal scan may not reveal renal hypoplasia since comparison of relative function may be made between two abnormal kidneys. We tested the hypothesis that ultrasonography in the neonatal period may be accurate and complementary to renal scan in detecting renal hypoplasia at birth. MATERIALS AND METHODS: Twenty-six infants who presented in the antenatal period with history of hydronephrosis and were noted to have neonatal vesicoureteral reflux postnatally were studied retrospectively. They had all been treated by a prospective protocol that included renal ultrasound and renal scans in the first 6 weeks of life. All had been placed on prophylactic antibiotics and had no urinary tract infection. Multiple sonographic parameters were analyzed including kidney length, echogenicity, calyceal blunting, parenchymal thinning and focal scars. We correlated the renal morphology on ultrasound, the renal function on renal scan and the degree of reflux seen on VCUG. RESULTS: VCUG showed reflux in 44 renal units, grade of reflux was: I (2), II (7), III (12), IV (8), and V (15). A variety of nucleides were used including DMSA in 15, DTPA in 6 and MAGIII in 5. Renal scans identified global hypoplasia without focal scars (differential function less than 40%) in 10 of 44 refluxing renal units grades I (1), III (2), IV (4), and V (3). The sonographic finding of decreased renal length (<50th percentile for age) was present in 14 refluxing units of 44 refluxing renal units, grade I (1), grade III (4 bilaterally in 1), grade IV (4 bilaterally in 1), and grade V (5 bilaterally in 2). The sonographic finding of decreased renal length (<50th percentile) correlated strongly with renal hypoplasia on renal scans in refluxing renal units (p value <.005, sensitivity 80% and specificity 82%, positive predictive value 57%, and negative predictive value 93%). CONCLUSION: Postnatal ultrasonography is a reliable measure of gross renal parenchyma, and in the presence of vesicoureteral reflux correlates with renal scintilligraphy. In addition, for cases of bilateral neonatal vesicoureteral reflux, ultrasound and renal scan are complimentary, each being able to detect the abnormalities that might be missed by the other.

[Pathologic development of the kidney]. / Pathologische Entwicklung der Nieren.

In the absence of firmly established views on the development of nephropathy, we describe in this paper the embryogenetic and clinical aspects of kidney disease. Congenital reductive nephropathy always arises in the ureteral bud and is determined by two factors, endogenous dysplasia and endogenous obstruction. The nine well-known patterns of disease that may result are described herein. The most important starting points are as follows: (a) A dysplastically disorganized and hence refluxive trigone of the bladder induces, via pyramidal-medullary deficiencies, a defect of the metanephros and thus what we term reflux nephropathy (III-V). BU and PN may supervene postnatally. (b) Similarly, obstruction of the ureteral outlet in the first trimester induces dysplastic ascending nephropathy. (c) The same obstruction beginning in the second trimester induces nondysplastic, purely obstructive nephropathy, characterized by glomerular hypogenesis and hemo-obliterative cirrhosis which varies considerably from stage to stage and from case to case and may go as far as complete loss of the parenchyma. (d) Obstruction of the pyeloureteral junction, occurring late in the embryonic phase and originating outside the urinary system, provides the clearest example of fully developed nondysplastic reductive nephropathy. The lesional process may come to a halt at any time. (e) Coincidence of early embryonic dysplastic-refluxive nephropathy and late embryonic infravesical obstruction (with no causal link) accounts for half the morbidity from valvular disease. The other half results from simple nondysplastic obstruction.

[The physiopathology of vesicoureteral reflux (author's transl)]. / Die Physiopathologie des primären vesikoureteralen Refluxes.

The tightness of the ureterovesical junction depends on all the structures composing the terminal and intra-mural ureter. The muscular, collagenic, and elastic fibers of the ureter constitute a mesh net which is stretched during bladder distention and closes the ureteric orifice as a valve. Congenital vesico-ureteric reflux results from a primary structural insufficiency of the terminal ureter or insufficiency of the bladder wall backing. As embryology shows primary reflux can be caused by a high ectopic implantation of the ureter. It results from the development of an ureteric bud appearing in a lower than normal position on the Wolffian duct. This results in a higher and more lateral opening of the ureteric orifice in the bladder which leads to a shorter intra-mural tunnel predisposing it to reflux. Secondary or acquired refluxes are associated with chronic obstructions (i.e. neurogenic bladder--lower urinary tract obstruction), and inflammatory lesions. Their pathogeneses are described and discussed. The maturation of the ureterovesical junction is considered a mechanism involving a reduced likelihood of secondary reflux. It may also, during the first years of life, palliate some minimal structural deficiencies of the intra-mural ureter, but growth and development are unlikely to normalize an ureter presenting at birth with a severe constitutional anomaly.

Vesicoureteral reflux in pediatric age: where are we today?

Although the diagnosis of vesicoureteral reflux and of reflux nephropathy is a well-established and shared procedure, its treatment nowadays is still very controversial. New developments on the knowledge of pathophysiology of renal damage associated to reflux opened the way towards a different diagnostic work-up and different therapeutic approaches. Recently, the "top-down" diagnostic approach has gained wider interest, versus the "down-top" protocol. The attention has recently focused on the renal parenchyma damage and less interest has been given to the presence and the radiological degree of vesicoureteral reflux. The review criteria were based on an in-depth search of references conducted on PubMed, using the terms "vesicoureteral reflux", "children", "incidence", "etiology", "diagnosis", "treatment" and "outcomes". The selection of the papers cited in this review was influenced by the content and the relevance to the points focused in the article.Conservative approaches include no treatment option with watchful waiting, long-term antibiotic prophylaxis and bladder rehabilitation. The operative treatment consists of endoscopic, open, laparoscopic and robotic procedures to stop the refluxing ureter.No final consensus has been achieved in literature yet, and further studies are necessary in order to better define the subset of children at risk of developing progression of renal damage.This review aims to clarify the diagnostic management and the urological-nephrological treatment of reflux in pediatric age, on the basis of a review of the best-published evidence.

Vesicoureteral reflux and other urinary tract malformations in mice compound heterozygous for Pax2 and Emx2.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. This disease group includes a spectrum of urinary tract defects including vesicoureteral reflux, duplex kidneys and other developmental defects that can be found alone or in combination. To identify new regulators of CAKUT, we tested the genetic cooperativity between several key regulators of urogenital system development in mice. We found a high incidence of urinary tract anomalies in Pax2;Emx2 compound heterozygous mice that are not found in single heterozygous mice. Pax2⁺/⁻;Emx2⁺/⁻ mice harbor duplex systems associated with urinary tract obstruction, bifid ureter and a high penetrance of vesicoureteral reflux. Remarkably, most compound heterozygous mice refluxed at low intravesical pressure. Early analysis of Pax2⁺/⁻;Emx2⁺/⁻ embryos point to ureter budding defects as the primary cause of urinary tract anomalies. We additionally establish Pax2 as a direct regulator of Emx2 expression in the Wolffian duct. Together, these results identify a haploinsufficient genetic combination resulting in CAKUT-like phenotype, including a high sensitivity to vesicoureteral reflux. As both genes are located on human chromosome 10q, which is lost in a proportion of VUR patients, these findings may help understand VUR and CAKUT in humans.

Noninvasive assessment of antenatal hydronephrosis in mice reveals a critical role for Robo2 in maintaining anti-reflux mechanism.

Antenatal hydronephrosis and vesicoureteral reflux (VUR) are common renal tract birth defects. We recently showed that disruption of the Robo2 gene is associated with VUR in humans and antenatal hydronephrosis in knockout mice. However, the natural history, causal relationship and developmental origins of these clinical conditions remain largely unclear. Although the hydronephrosis phenotype in Robo2 knockout mice has been attributed to the coexistence of ureteral reflux and obstruction in the same mice, this hypothesis has not been tested experimentally. Here we used noninvasive high-resolution micro-ultrasonography and pathological analysis to follow the progression of antenatal hydronephrosis in individual Robo2-deficient mice from embryo to adulthood. We found that hydronephrosis progressed continuously after birth with no spontaneous resolution. With the use of a microbubble ultrasound contrast agent and ultrasound-guided percutaneous aspiration, we demonstrated that antenatal hydronephrosis in Robo2-deficient mice is caused by high-grade VUR resulting from a dilated and incompetent ureterovesical junction rather than ureteral obstruction. We further documented Robo2 expression around the developing ureterovesical junction and identified early dilatation of ureteral orifice structures as a potential fetal origin of antenatal hydronephrosis and VUR. Our results thus demonstrate that Robo2 is crucial for the formation of a normal ureteral orifice and for the maintenance of an effective anti-reflux mechanism. This study also establishes a reproducible genetic mouse model of progressive antenatal hydronephrosis and primary high-grade VUR.