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.

A single-center cohort of Canadian children with VUR reveals renal phenotypes important for genetic studies.

BACKGROUND: Many genes and loci have been reported in genetic studies of primary vesicoureteral reflux (VUR), but few have been reproduced in independent cohorts, perhaps because of phenotype heterogeneity. We phenotyped children with VUR who attended urology clinics so we could establish criteria to stratify patients based on the presence or absence of a renal malformation. METHODS: History, chart review, and DNA were obtained for 200 children with VUR from 189 families to determine the grade of VUR, the mode of presentation, and the family history for each child. Kidney length measured on ultrasound (US) and technetium dimercaptosuccinic acid (DMSA) scans at the time of VUR diagnosis were used to establish the presence of a concurrent renal malformation and identify the presence of renal scarring. RESULTS: There was an even distribution of girls and boys, and most patients were diagnosed following a urinary tract infection (UTI). Thirty-four percent of the children had severe VUR, and 25 % had undergone surgical correction. VUR is highly heritable, with 15 % of the families reporting multiple affected members. Most patients had normally formed kidneys as determined by US and DMSA imaging. Of the 93 patients who underwent DMSA imaging, 17 (18 %) showed scarring, 2 (2 %) showed scarring and diffuse reduction in uptake, and 13 (14 %) showed an isolated diffuse reduction in uptake. CONCLUSION: Prospective long-term studies of patients with primary VUR combined with renal phenotyping using US and DMSA imaging are needed to establish the presence of a renal malformation. The majority of patients in our study had no renal malformation. This cohort is a new resource for genetic studies of children with primary VUR.

T-cell factor/ß-catenin activity is suppressed in two different models of autosomal dominant polycystic kidney disease.

During murine kidney development, canonical WNT signaling is highly active in tubules until about embryonic days E16-E18. At this time, ß-catenin transcriptional activity is progressively restricted to the nephrogenic zone. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease (ADPKD), and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signaling activity. Several in vitro studies have found a link between cilial signaling and ß-catenin regulation, suggesting that aberrant activity might contribute to the cystic phenotype. To study this, we crossed T-cell factor (TCF)/ß-catenin-lacZ reporter mice with mice having Pkd1 or Pkd2 mutations and found that there was no ß-galactosidase staining in cells lining the renal cysts. Thus, suppression of canonical WNT activity, defined by the TCF/ß-catenin-lacZ reporter, is normal in these two different models of polycystic kidney disease. Hence, excessive ß-catenin transcriptional activity may not contribute to cystogenesis in these models of ADPKD.

Vesico-ureteric reflux: using mouse models to understand a common congenital urinary tract defect.

Vesico-ureteric reflux (VUR) is a common congenital urinary tract defect in which urine flows retrogradely from the bladder to the kidneys because of an abnormally formed uretero-vesical junction. It is associated with recurrent urinary tract infections, renal hypo/dysplasia, reflux nephropathy, hypertension, and end-stage renal disease. In humans, VUR is genetically and phenotypically heterogeneous, encompassing diverse renal and urinary tract phenotypes. To understand the significance of these phenotypes, we and others have used the mouse as a model organism and this has led to the identification of new candidate genes. Through careful phenotypic analysis of these models, a new understanding of the genetics and biology of VUR is now underway.

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.

High incidence of vesicoureteral reflux in mice with Fgfr2 deletion in kidney mesenchyma.

PURPOSE: Mice with Fgfr2 conditional deletion in metanephric mesenchyma (Fgfr2(Mes-/-)) have ureteral bud induction abnormalities. We determined whether Fgfr2(Mes-/-) mutants developed abnormally positioned ureters predisposing to vesicoureteral reflux. MATERIALS AND METHODS: We measured common nephric duct length and assayed for apoptosis in embryonic day 11.5 mice. We performed 3-dimensional reconstruction of, and real-time polymerase chain reaction and whole mount in situ hybridization for Fgfr2 in urinary tracts in embryonic day 15.5 embryos. We also performed cystograms followed by 3-dimensional reconstruction in postnatal animals. RESULTS: Compared with controls Fgfr2(Mes-/-) embryos had increased common nephric duct length with no difference in apoptosis, indicating cranially displaced ureteral buds. Three-dimensional reconstruction at embryonic day 15.5 showed low ureteral insertion into the bladder near the bladder neck in Fgfr2(Mes-/-) mice. Postnatal Fgfr2(Mes-/-) mutants had a high rate of vesicoureteral reflux compared with controls (47.4% vs 4.0%, p = 0.00006). In postnatal mutants with unilateral reflux the refluxing ureters inserted closer to the bladder neck than nonrefluxing ureters. External ureteral insertional angles at the outer bladder wall formed by the ureteral insertion points and the bladder neck were greater in mutant refluxing ureters than in contralateral nonrefluxing ureters or control ureters. At embryonic day 15.5 Fgfr2 was decreased in Fgfr2(Mes-/-) kidneys compared with that in controls but not statistically different in ureters or bladders. CONCLUSIONS: Fgfr2(Mes-/-) mice have ureteral induction abnormalities associated with abnormal ureteral insertion in the bladder and subsequent vesicoureteral reflux, consistent with the Mackie and Stephens hypothesis.

An Official American Thoracic Society Workshop Report: Translational Research in Rare Respiratory Diseases.

Rare respiratory diseases (RRDs) are a heterogeneous group of disorders that collectively represent a significant health care burden. In recent years, strong advocacy and policy initiatives have led to advances in the implementation of research and clinical care for rare diseases. The development of specialized centers and research networks has facilitated support for affected individuals as well as emerging programs in basic, translational, and clinical research. In selected RRDs, subsequent gains in knowledge have informed the development of targeted therapies and effective diagnostic tests, but many gaps persist. There was therefore a desire to identify the elements contributing to an effective translational research program in RRDs. To this end, a workshop was convened in October 2015 with a focus on the implementation of effective transnational research networks and collaborations aimed at developing novel diagnostic and therapeutic tools. Key elements included an emphasis on molecular pathogenesis, the continuing engagement of patient advocacy groups and policy makers, the effective use of preclinical models in the translational research pipeline, and the detailed phenotyping of patient cohorts. During the course of the workshop, current logistical and knowledge gaps were identified, and new solutions or opportunities were highlighted.

Interplay between vesicoureteric reflux and kidney infection in the development of reflux nephropathy in mice.

Vesicoureteric reflux (VUR) is a common congenital defect of the urinary tract that is usually discovered after a child develops a urinary tract infection. It is associated with reflux nephropathy, a renal lesion characterized by the presence of chronic tubulointersitial inflammation and fibrosis. Most patients are diagnosed with reflux nephropathy after one or more febrile urinary tract infections, suggesting a potential role for infection in its development. We have recently shown that the C3H mouse has a 100% incidence of VUR. Here, we evaluate the roles of VUR and uropathogenic Escherichia coli infection in the development of reflux nephropathy in the C3H mouse. We find that VUR in combination with sustained kidney infection is crucial to the development of reflux nephropathy, whereas sterile reflux alone fails to induce reflux nephropathy. A single bout of kidney infection without reflux fails to induce reflux nephropathy. The host immune response to infection was examined in two refluxing C3H substrains, HeN and HeJ. HeJ mice, which have a defect in innate immunity and bacterial clearance, demonstrate more significant renal inflammation and reflux nephropathy compared with HeN mice. These studies demonstrate the crucial synergy between VUR, sustained kidney infection and the host immune response in the development of reflux nephropathy in a mouse model of VUR.

Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) encompass a spectrum of kidney and urinary tract disorders. Here, we describe two assays that can be used to determine if a mouse has vesicoureteric reflux (VUR) or urinary tract obstruction, two urinary tract defects observed in CAKUT. To test for VUR, dye is injected into the mouse bladder and then monitored to determine if it passes retrogradely from the bladder towards the kidneys, indicating the presence of VUR. To test for urinary tract obstruction, the renal pelvis is microinjected with dye and its passage along the urinary tract is monitored to determine if there is evidence of impaired flow along the tract. These methods will facilitate the analysis of CAKUT phenotypes in the mouse.

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.

The relationship between nephron number, kidney size and body weight in two inbred mouse strains.

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.

Gene discovery and vesicoureteric reflux.

Vesicoureteric reflux (VUR) is a congenital urinary tract defect caused by abnormal insertion of the ureter within the bladder wall. This leads to a defective ureterovesical junction in which urine flows retrogradely from the bladder to the kidneys. Although VUR is associated with recurrent urinary tract infections, renal malformations, hypertension, and reflux nephropathy, its relationship to each of these clinical entities is poorly understood. Mutations in genes expressed by the developing kidney and urinary tract can cause VUR in mice, and some of these same genes have been identified in humans with VUR. By discovering the genes that are associated with VUR, new hypotheses will be generated such that, eventually, the relationship between VUR and its complications will be understood.

Vesico-ureteric reflux and urinary tract development in the Pax2 1Neu+/- mouse.

Vesico-ureteric reflux (VUR) is a urinary tract abnormality that affects roughly one-third of patients with renal-coloboma syndrome, an autosomal dominant condition caused by a mutation in PAX2. Here, we report that a mouse model with an identical mutation, the Pax2 1Neu+/- mouse, has a 30% incidence of VUR. In VUR, urine flows retrogradely from the bladder to the ureter and is associated with urinary tract infections, hypertension, and renal failure. The propensity to reflux in the Pax2 1Neu+/- mouse is correlated with a shortened intravesical ureter that has lost its oblique angle of entry into the bladder wall compared with wild-type mice. Normally, the kidney and urinary tract develop from the ureteric bud, which grows from a predetermined position on the mesonephric duct. In Pax2 1Neu+/- mice, this position is shifted caudally while surrounding metanephric mesenchyme markers remain unaffected. Mutant offspring from crosses between Pax2 1Neu+/- and Hoxb7/GFP+/- mice have delayed union of the ureter with the bladder and delayed separation of the ureter from the mesonephric duct. These events are not caused by a change in apoptosis within the developing urinary tract. Our results provide the first evidence that VUR may arise from a delay in urinary tract maturation and an explanation for the clinical observation that VUR resolves over time in some affected children.