Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.     

Molecular pathology of murine ureteritis causing obstructive uropathy with hydronephrosis

Primary causes of urinary tract obstruction that induces urine retention and results in hydronephrosis include uroliths, inflammation, and tumors. In this study, we analyzed the molecular pathology of ureteritis causing hydronephrosis in laboratory rodents.F2 progenies of C57BL/6 and DBA/2 mice were studied histopathologically and by comprehensive gene expression analysis of their ureters. Incidence of hydronephrosis was approximately 5% in F2 progenies. Histopathologically, this hydronephrosis was caused by stenosis of the proximal ureter, which showed fibrosis and papillary malformations of the proliferative epithelium with infiltrations of B-cell-dominated lymphocytes. Additionally, CD16-positive large granular leukocytes and eosinophils infiltrated from the ureteral mucosa to the muscular layer. Eosinophilic crystals were characteristically observed in the lumen of the ureter and the cytoplasm of large granular leukocytes, eosinophils, and transitional epithelial cells. Comprehensive gene profiling revealed remarkably elevated expression of genes associated with hyperimmune responses through activation of B cells in diseased ureters. Furthermore, diseased ureters showed dramatically higher gene expression of chitinase 3-like 3, known as Ym1, which is associated with formation both of adenomas in the transitional epithelium and of eosinophilic crystals in inflammatory conditions. The Ym1 protein was mainly localized to the cytoplasm of the transitional epithelium, infiltrated cells, and eosinophilic crystals in diseased ureters.We determined that the primary cause of hydronephrosis in F2 mice was ureteritis mediated by the local hyperimmune response with malformation of the transitional epithelium. Our data provide a novel molecular pathogenesis for elucidating causes of aseptic inflammation in human upper urinary tracts.     

Effects of TCDD on embryonic ureteric epithelial EGF receptor expression and cell proliferation

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing hydronephrosis and cleft palate. Because of the long half-life of TCDD, the urinary tract is exposed throughout development after a single dose on gestation day (GD) 10 or earlier. TCDD-induced hydronephrosis is a consequence of occlusion of the ureter by epithelial cells. Since embryonic growth factors and the epidermal growth factor (EGF) receptor are probably involved in regulation of embryonic cell proliferation, this study examines the effects of TCDD on expression of EGF receptors and proliferation of ureteric epithelial cells in vivo and in culture. After exposure to TCDD by gavage (12, 24, or 30 micrograms/kg on GD 10; 6 or 24 micrograms/kg on GD 12) the mean cell depth of the ureteric and bladder epithelia was increased. EGF receptors were detected immunohistochemically in sectioned urinary tracts. The expression of receptors decreased with advancing development in control ureteric epithelia. However, after TCDD exposure the level of EGF receptors failed to decline. The incorporation of 3H-TdR was observed in sections by autoradiography, and after exposure to TCDD more epithelial cells showed incorporation than was apparent in controls. Transmission electron microscopy (TEM) of embryonic ureters from fetuses exposed to TCDD in vivo showed no cytotoxicity in basal cells and the cells remained undifferentiated, as in controls. Ureters taken from GD 12 embryos and cultured with 1 x 10(-10)M TCDD showed ureteric epithelial hyperplasia without cytotoxicity, but at 1 x 10(-8)M TCDD evidence of cytotoxicity was observed by TEM. The levels of TCDD found in fetuses after in vivo exposure (204-307 pg/fetus, with 1-2 pg in the urinary tract) compare well with the in vitro level (32 pg/ml), which was most effective in producing hyperplasia of the epithelial cells. The present study correlates a TCDD-induced increase in cell depth with altered regulation of EGF receptors and excessive proliferation, both in vivo and in cultured embryonic ureters.     

Fstl1 antagonizes BMP signaling and regulates ureter development

Bone morphogenetic protein (BMP) signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1), encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH) signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1(-/-) ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.     

Obstructive uropathy secondary to ureteroinguinal herniation

Herniation of the ureter occurs infrequently in a sliding inguinal hernia. Massive herniation may cause ureteral obstruction leading to hydronephrosis. Computed tomography can demonstrate both ureteral herniation and associated hydronephrosis.     

Effect of maternal restraint stress on fetal development of ICR mice.

The present study was conducted to elucidate the susceptibility of embryos and fetuses at different gestational stages to the maternal stress in mice. Groups of pregnant ICR mice were subjected to daily 12-h restraint stress, taped in the supine position on a plastic board, on gestational days (GD) 1-4, 5-8, 9-12 and 13-16, respectively. Caesarean sections were performed on gestational day 18, and the fetuses were weighed and examined for morphological defects. During the daily restraint for 4 days, the maternal body weights markedly decreased. Although the body weights recovered gradually after termination of the stress, the recovery was not full until the final stage of pregnancy. Interestingly, restraint stress caused growth retardation of the fetuses, leading to a significant decrease in their body weights, and increased early and late resorptions of embryos and fetuses according to the stress periods. Although the preceding (GD1-4) and concurrent (GD5-8) stresses did not affect embryonic implantation, restraint stress on GD9-12 caused cleft palate. Whereas vertebral abnormalities, mainly bipartite ossification, were observed only in animals stressed on GD5-8, abnormalities of sternebrae, exhibiting asymmetric or bipartite ossification, were enhanced by the stress at all of the gestational stages. On the other hand, the incidence of other malformations including renal malposition and costal abnormalities was not increased by stress at any of the 4 stages. Taken together, the results suggest that intensive restraint stress influences the maternal body weight resulting in growth retardation and increased mortality of embryos and fetuses, in addition to gestational stage-specific ventricular dilatation, cleft palate and sternal abnormalities.     

Incontinence in a child with a duplex kidney: case report

Persistent incontinence after toilet training in young girls and urinary tract infections or epididymitis in prepubertal boys should raise suspicion of an ectopic ureter. This often occurs in the context of duplication of kidney structures or other parenchymal abnormalities. The presence or absence of reflux affects surgical treatment, which may consist of ureteral reimplantation, ureteroureterostomy, and/or upper pole nephrectomy.     

Interstitial cell of Cajal-like cells in the upper urinary tract

Autorhythmicity in the upper urinary tract (UUT) has long been considered to arise in specialized atypical smooth muscle cells (SMC) predominately situated in the most proximal regions of the pyeloureteric system. These atypical SMC pacemakers have been thought to trigger adjacent electrically-quiescent typical SMC to fire action potentials which allow an influx of Ca2+ and the generation of muscle contraction. More recently, the presence of cells with many of the morphological, electrical and immunohistochemical characteristics of interstitial cells of Cajal (ICC), the pacemaker cells of the gastrointestinal tract, have been located in many regions of both the upper and lower urinary tract. This article reviews the evidence from the literature and from our laboratory supporting a role of both atypical SMC and ICC-like cells in the initiation and propagation of pyeloureteric peristalsis in the UUT. We propose a new model in which there are 2 populations of pacemaker cells, high frequency atypical SMC and lower frequency ICC-like cells, both of which can drive electrically-quiescent typical SMC. The relative presence of these 2 populations of pacemaker cells and the relatively-long refractoriness of typical SMC determines the decreasing frequency of contraction with distance from the renal fornix. In the absence of the proximal pacemaker drive from atypical SMC after pyeloureteral/ureteral obstruction or surgery, ICC-like cell pacemaking provides a compensatory mechanism allowing the ureter to maintain rudimentary peristaltic waves and movement of urine from the pyelon towards the bladder.     

The hedgehog signal induced modulation of bone morphogenetic protein signaling: an essential signaling relay for urinary tract morphogenesis

Background

Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases.

Methodology/Principal Findings

To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh^−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells.

Conclusions/Significance

This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh-responsive mesenchymal Bmp signaling maintains the population of peri-cloacal mesenchyme cells, which is essential for the development of the ureter and the upper urinary tract.     

A reproductive screening test of goldenseal

BACKGROUND: Goldenseal (Hydrastis canadensis L) is a multi-purpose herb (Hobbs, 1990: Pharm Hist 32:79-82) widely used for its antibiotic properties. It is traditionally contraindicated in pregnancy based on in vivo data but this contraindication has not been confirmed by conventional studies that have been peer reviewed. METHODS: Female rats were dosed by gavage using 65 times the human dose of goldenseal daily on either gestation days (GD) 1-8 or GD 8-15. Controls received an equivalent dose of ethanol. On GD 20, fetuses were weighed and examined for signs of external, internal, or skeletal malformations. Rat fetuses were also explanted on GD 10.5 and cultured with decreasing concentrations of goldenseal for 26 hr. Embryos were examined for growth retardation and malformations. RESULTS: There was no increase in pre- or post-implantation losses. There was no increase in fetal body weight in fetuses exposed to goldenseal. There was no difference in incidence of external or internal malformations. Goldenseal induced toxicity when GD10.5 embryos were cultured for 26 hr in rat serum to which extract was added. CONCLUSIONS: It is likely that poor absorption of goldenseal from the small intestine could explain the discrepancy between the in vivo and in vitro results. It is unlikely that serum concentration from oral treatment could attain the LOEL achieved in vitro. The contrasting results highlight the continuing importance of in vivo work and the necessity of pharmacokinetic data when interpreting in vitro data. The data suggest that goldenseal, at the prescribed human dose, is unlikely to be absorbed to an extent to be unsafe to use in pregnancy despite the apparent cytotoxic effects in vitro. However, these results indicate that pharmacokinetic studies are required to confirm this conclusion.     

Effects of in vivo exposure of pregnant hamsters to glucose. 1. Abnormalities in LVG strain fetuses following intermittent multiple treatments with two isomers

The increased frequency of congenital malformations including caudal regression syndrome, in infants of women with insulin-dependent diabetes mellitus is well documented. Most of the related animal research has involved the in vitro embryo culturing methodology. This study involved the alternative in vivo approach in order to determine the effects of treatment of pregnant hamsters with the D- and L-isomers of glucose at five times just before and during the period of embryonic organogenesis on maternal blood glucose levels and the rates and types of fetal abnormalities. One group of animals was injected with 5 doses (4 g/kg each) of D-glucose, i.e., on gestation day (D) 6, 3 PM; D7, 8 AM and 3 PM; D8, 8 AM and 3 PM. Two other groups were treated the same way but with L-glucose (4 g/kg per dose) and water (10 ml/kg per dose), respectively. The D-glucose treatment produced alternating periods of hyperglycemia and normoglycemia in the pregnant hamsters, enlarged placentae and fetuses with small urinary bladders, microphthalmia and skeletal abnormalities of the sternum, caudal vertebrae, pelvic bones, and femora. The L-glucose treatment did not produce changes in maternal blood D-glucose levels but did produce fetuses with small urinary bladders, microphthalmia and abnormal ossification limited to the manubrium. Several interpretations of the D-glucose-induced fetal abnormalities involving the vertebrae, proximal hindlimb bones and urinary bladders are discussed, including the consideration that this cluster has interesting similarities to the spectrum of skeletal and soft tissue abnormalities of human diabetes-related caudal regression syndrome.     

输尿管子宫内膜异位症伴肾积水1 例报告并文献复习

目的:探讨子宫内膜异位症累及输尿管的诊断和治疗方法。方法:术前诊断为右侧输尿管下段占位病变伴右肾积水的42岁女性患者,行下腹正中切口,探查右侧输尿管开口处可见淡黄色息肉样病变,突入膀胱,输尿管下段增粗并全程扩张积水,行输尿管下段并膀胱袖式切除,输尿管膀胱再植术。术后病理报告为输尿管子宫内膜异位症。结果:术后复查B超示右肾积水较术前恢复,术后予抑那通3.75mg/28d,随访6个月未见复发。结论:对于输尿管占位并上尿路积水的女性患者,除考虑肿瘤外还应考虑子宫内膜异位症可能。手术联合内分泌治疗是治疗输尿管子宫内膜异位伴肾积水的有效方法。     

Urine cytology of localized primary amyloidosis of the ureter: a case report

BACKGROUND: Morphologic findings of amyloid in urine cytology material have rarely been reported because amyloidosis of the urinary tract is a relatively uncommon disorder. We present a case of primary amyloidosis of the ureter, including catheterized urine cytologicfindings. CASE: A 78-year-old man had pollakiuria and dysuria for 5 years before admission after transurethral resection of the prostate. Clinical examination revealed left hydronephrosis and stricture of the lower part of the left ureter, and a malignant ureteral tumor was suspected clinically. In catheterized urine cytology, many clusters of epithelial cells, inflammatory cells and abundant, amorphous, waxy material were observed. The amorphous material stained light green by the Papanicolaou method and positive with direct fast scarlet (DFS), showing yellow-green birefringence under polarized light. Positivity with DFS staining was not affected by treatment with potassium permanganate. Immunocytochemically the material was AL-type amyloid protein. Atypia were absent from epithelial cells. The patient had no history of diseases that could cause secondary amyloidosis. The present case was considered to be primary amyloidosis localized to the left ureter because no particular morphologic change in the epithelial cells of the urinary tract was observed. CONCLUSION: Amyloid can be present in urine and should not be overlooked or confused with tumor diathesis when a malignant tumor is suspected clinically.     

Preliminary screening study of reproductive outcomes after exposure to yarrow in the pregnant rat

BACKGROUND: Yarrow (Achillea millefolium) is used for the treatment of diarrhea, as a urinary antiseptic, hypotensive, and diuretic. Yarrow also has traditionally been used as an abortifacient, emmenagogue, contraceptive, and for stimulating uterine contractions. For this reason, it is contra‐indicated for use in pregnancy. There has, however, been little scientific research carried out to either confirm or refute this recommendation. METHODS: Female rats were dosed, orally by gavage using 56 times the human dose of yarrow daily on either gestation days (GD) 1–8 or GD 8–15. Two groups of controls were included; the first received water and the second received an equivalent dose of ethanol to that found in the yarrow preparation over the two gestation periods. On GD 20, rats were sacrificed, placentae were weighed, and corpora lutea counted. The fetuses were weighed and examined for signs of external, internal or skeletal malformations. RESULTS: The dose used was not materno‐toxic. There was no increase in pre‐ or post‐implantation losses suggesting that yarrow was neither an abortifacient nor a contraceptive. Placental weights were increased in rats treated with yarrow on GD 8–15 compared to water and ethanol controls and on GD 1–8 compared to water control fetuses. Body weight was reduced in fetuses exposed to yarrow on GD 8–15 compared to water control fetuses. There was no difference in incidence of external or internal malformations. CONCLUSIONS: In the present study it was found that yarrow, when administered to rats at 56 times the human dose, was associated with reduced fetal weight and increased placental weight. In the absence of a no observable effect level for these variables it must be concluded that the consumption of yarrow is contraindicated during pregnancy until further investigations have been carried out. Birth Defects Res B 68:416–420, 2003. © 2003 Wiley‐Liss, Inc.     

胎羊单侧输尿管梗阻模型的建立

目的建立胎羊单侧输尿管梗阻的动物模型,探讨其病理、影像学特点。方法取12只单胎妊娠75-85 d的健康山羊,采用宫内手术的方法造成胎羊左侧输尿管不完全梗阻。对羔羊进行影像、病理学研究。结果12只孕羊中有3只流产;有9只孕羊顺产羔羊。超声检查：梗阻后的第3周内胎羊左肾显著增大、积水及实质变薄。放射学检查：羔羊左肾积水并且功能受损害。病理学检查：左肾肾小球数目减少,肾小管扩张明显,未见肾发育不良。结论对山羊单胎妊娠中期胎羊进行宫内手术建立胎羊单侧输尿管梗阻的动物模型是可行的,该模型能很好地模拟肾盂输尿管连接部梗阻所致的胎儿肾积水。     

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.     

The development of the bladder trigone, the center of the anti-reflux mechanism

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development.