Renal abnormalities and their developmental origin

Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 1 out of 500 newborns, and constitute approximately 20-30% of all anomalies identified in the prenatal period. CAKUT has a major role in renal failure, and there is increasing evidence that certain abnormalities predispose to the development of hypertension and cardiovascular disease in adult life. Moreover, defects in nephron formation can predispose to Wilms tumour, the most frequent solid tumour in children. To understand the basis of human renal diseases, it is essential to consider how the kidney develops.     

Genetic Basis of Ureterocele

Congenital anomalies of the kidney and urinary tract (CAKUT) form a group of heterogeneous disorders that affect the kidneys, ureters and bladder, with frequent asynchronous presentations and multiple CAKUT associations in the same individual. Urinary tract formation is a complex process, dependent of the interaction of multiple genes and their sub-product. The same genic alterations can lead to different molecular expressions and different morphological anomalies. The ureterocele is a cystic dilation of the distal intramural ureter, resulting in obstruction of urine flow, dilation of the ureter and renal pelvis and loss of renal function. Two key steps in the urinary tract ontogenesis may be related to ureterocele development: formation and migration of the ureteric bud and its incorporation in the bladder. This review aims to describe the morphological, cellular and biochemical steps, as well as the genes involved in the occurrence of this anomaly.     

Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.     

Development of the kidney medulla

The mature renal medulla, the inner part of the kidney, consists of the medullary collecting ducts, loops of Henle, vasa recta and the interstitium. The unique spatial arrangement of these components is essential for the regulation of urine concentration and other specialized kidney functions. Thus, the proper and timely assembly of medulla constituents is a crucial morphogenetic event leading to the formation of a functioning metanephric kidney. Mechanisms that direct renal medulla formation are poorly understood. This review describes the current understanding of the key molecular and cellular mechanisms underlying morphological aspects of medulla formation. Given that hypoplasia of the renal medulla is a common manifestation of congenital obstructive nephropathy and other types of congenital anomalies of the kidney and urinary tract (CAKUT), better understanding of how disruptions in medulla formation are linked to CAKUT will enable improved diagnosis, treatment and prevention of CAKUT and their associated morbidity.     

Development of the kidney medulla

The mature renal medulla, the inner part of the kidney, consists of the medullary collecting ducts, loops of Henle, vasa recta and the interstitium. The unique spatial arrangement of these components is essential for the regulation of urine concentration and other specialized kidney functions. Thus, the proper and timely assembly of medulla constituents is a crucial morphogenetic event leading to the formation of a functioning metanephric kidney. Mechanisms that direct renal medulla formation are poorly understood. This review describes the current understanding of the key molecular and cellular mechanisms underlying morphological aspects of medulla formation. Given that hypoplasia of the renal medulla is a common manifestation of congenital obstructive nephropathy and other types of congenital anomalies of the kidney and urinary tract (CAKUT), better understanding of how disruptions in medulla formation are linked to CAKUT will enable improved diagnosis, treatment and prevention of CAKUT and their associated morbidity.     

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 many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret''s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret''s signaling specificity and intergenic interactions that confer normal urinary system development.Key words: RET, GDNF, kidney, RTK, CAKUT, branching morphogenesis, ureter     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfrα1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfrα1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.     

Genetic Etiology of Renal Agenesis: Fine Mapping of Renag1 and Identification of Kit as the Candidate Functional Gene

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.     

Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux

Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR.     

Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice

Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCγ, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT.     

Genetics of Vesicoureteral Reflux

Vesicoureteral reflux (VUR) is the retrograde passage of urine from the bladder to the upper urinary tract. It is the most common congenital urological anomaly affecting 1-2% of children and 30-40% of patients with urinary tract infections. VUR is a major risk factor for pyelonephritic scarring and chronic renal failure in children. It is the result of a shortened intravesical ureter with an enlarged or malpositioned ureteric orifice. An ectopic embryonal ureteric budding development is implicated in the pathogenesis of VUR, which is a complex genetic developmental disorder. Many genes are involved in the ureteric budding formation and subsequently in the urinary tract and kidney development. Previous studies demonstrate an heterogeneous genetic pattern of VUR. In fact no single major locus or gene for primary VUR has been identified. It is likely that different forms of VUR with different genetic determinantes are present. Moreover genetic studies of syndromes with associated VUR have revealed several possible candidate genes involved in the pathogenesis of VUR and related urinary tract malformations. Mutations in genes essential for urinary tract morphogenesis are linked to numerous congenital syndromes, and in most of those VUR is a feature. The Authors provide an overview of the developmental processes leading to the VUR. The different genes and signaling pathways controlling the embryonal urinary tract development are analyzed. A better understanding of VUR genetic bases could improve the management of this condition in children.     

Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations

Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET-null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in six unrelated patients. A family with duplicated collecting system had a novel mutation, RET-R831Q, which showed markedly decreased GDNF-dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations was necessary to affect RET activity. Targeted whole-exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5?% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway.     

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.     

Abnormalities of the Urinary Tract in Association with Congenital Cardiovascular Disease

The incidence of urinary tract abnormalities in a group of infants and children with congenital cardiovascular disease was determined by assessing roentgenograms of the abdomen taken following angiocardiographic studies. The urinary tracts of 400 of these patients were so outlined, but in 50 others the tracts could not be outlined. In 21 of the latter the urinary tracts were assessed at autopsy.Of these 421 children, nine (approximately 2%) had serious urinary tract disease, and 15 (approximately 3.5%) had anomalies which were of no clinical significance.The incidence of four renal anomalies—bifid collecting system, rotated kidney, horsehoe kidney and ectopic kidney—was compared with that in another group of patients without cardiovascular disease. No significant difference was found in the incidence of these four anomalies in the two groups.     

Pathogenesis of ethanol-induced hydronephrosis and hydroureter as demonstrated following in vivo exposure of mouse embryos

Urinary tract abnormalities have been noted to occur in 10-27% of individuals diagnosed as having Fetal Alcohol Syndrome. Among a wide range of functional and structural abnormalities, renal agenesis/hypoplasia, hydronephrosis, and ureteropelvic obstruction feature most prominently. This study was designed to examine the pathogenesis of ethanol-induced urinary tract abnormalities in a mouse model. C57Bl/6J mice were acutely exposed to two doses of ethanol (2.9 g/kg IP) administered 4 hours apart beginning on gestational day (GD) 9, hour 4. This resulted in an incidence of 40.7% urinary tract anomalies among GD18 fetuses. With the exception of duplicate ureter, urinary tract abnormalities consisted exclusively of hydroureter/hydronephrosis. Examination of GD13-17 fetuses revealed that the first grossly detectable differences in the urinary tracts of control vs. affected specimens occurred on GD16 and initially only involved ureteral changes. Hydronephrosis was first detected on GD17. A contributing factor to the development of hydronephrosis appears to be the abnormal location of the ureterovesicle junction which commonly involves duplicate ureteral lumens with resultant functional obstruction to urine flow at the distal end of the ureter. Study of the early pathogenetic changes which appear to result in the urinary tract malformations observed involved utilization of scanning electron microscopy, vital dye (Nile blue sulphate) staining of whole embryos, and analysis of histological sections. These studies revealed that 12 hours following initial maternal ethanol exposure, embryos have excessive amounts of cell death localized in the region of the developing mesonephric duct just proximal to the cloaca. Also affected were premigratory neural crest cells located just proximal to the posterior neuropore. We conclude that excessive amounts of ethanol-induced cell death in these selectively vulnerable populations could account for the subsequently observed urinary tract malformations.     

Role of the angiotensin receptor in the development of the mammalian kidney and urinary tract

Recent gene targeting and pharmacological studies have shown that the renin-angiotensin system is essential for the development of the mammalian kidney and urinary tract system. We have generated mutant mice carrying a targeted null mutation of the angiotensin type 1 receptor (AT1) or type 2 receptor (AT2) gene. Analyses of these two mutant mice revealed that both mutants have a distinct phenotype in the kidney and urinary tract system. Thus, some AT2 null mutants (Agtr2 -/Y) show anomalies that mimic human congenital anomalies of the kidney and urinary tract as a result of a defect in the in utero organogenesis of the excretory system, while AT1 null mutants (Agtr1 -/-) exhibit hydronephrosis due to a defective development of the urinary peristaltic machinery during the perinatal period. In this review, we discuss chronologically from embryos to adults how the angiotensin receptors regulate the morphogenesis of the excretory system.