Obstructed Bifid Uretereric System Causing Unilateral Hydronephrosis

Bifid ureters are a common malformation of the urinary system. In clinical practice, hydronephrosis resulting from obstruction of such a system is rare. The authors present a case involving an 88-year-old man admitted to the hospital with symptoms of renal failure, where bifid ureters were found incidentally in a hydronephrotic kidney during an emergency nephrostomy. This had been missed on a previous CT scan, resulting in a unique therapeutic dilemma.Key words: Bifid ureters, Hydronephrosis, NephrostomyIn 1948, Nordmark described bifid ureters, along with bifid renal pelves, as the most common malformation of the urinary system. Later studies confirmed this, with duplicated ureters being found in 1 in 125 patients (0.8%) in whom postmortem examinations were carried out.^1,2 Often, these remain asymptomatic, and as a result, undiagnosed. In symptomatic patients, bifid ureteric systems are often found to be dilated as a consequence of blind-ending branches. Such cases have frequently been described in medical literature; however, the finding of hydronephrosis resulting from tumor obstructing the bifid ureter is relatively rare.We present a case report of hydronephrosis due to back pressure from obstruction of the ureteric orifice. The obstruction resulted in dilatation of both the moieties of the bifid ureters, causing both a diagnostic and treatment dilemma.     

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.     

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.     

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.     

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.     

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.     

Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.     

Hypothesis: A New Role for the Renin-Angiotensin System in Ureteric Bud Branching

Branching morphogenesis in the developing mammalian kidney involves growth and branching of the ureteric bud (UB), leading to formation of its daughter collecting ducts, calyces, pelvis and ureters. Even subtle defects in the efficiency and/or accuracy of this process have profound effects on the ultimate development of the kidney and result in congenital abnormalities of the kidney and urinary tract. This review summarizes current knowledge regarding a number of genes known to regulate UB development and emphasizes an emerging role for the renin-angiotensin system (RAS) in renal branching morphogenesis. Mutations in the genes encoding components of the RAS in mice cause renal papillary hypoplasia, hydronephrosis, and urinary concentrating defect. These findings imply that UB-derived epithelia are targets for angiotensin (ANG) II actions during metanephric kidney development. Here, it is proposed that papillary hypoplasia in RAS-deficient mice is secondary to an intrinsic defect in the development of the renal medulla. This hypothesis is based on the following observations: (a) UB and surrounding stroma express angiotensinogen (AGT) and ANG II AT₁ receptors in vivo; (b) ANG II stimulates UB cell process extension, branching and cord formation in collagen gel cultures in vitro; and (c) AT₁ blockade inhibits ANG II-induced UB cell branching. It is further postulated that ANG II is a novel stroma-derived factor involved in stroma/UB cross-talk which regulates UB branching morphogenesis.Key Words: kidney development, branching morphogenesis, renin-angiotensin, stromal mesenchyme, ureteric bud     

Smad4 Regulates Ureteral Smooth Muscle Cell Differentiation during Mouse Embryogenesis

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.     

Hypothesis: A New Role for the Renin-Angiotensin System in Ureteric Bud Branching

Branching morphogenesis in the developing mammalian kidney involves growth and branching of the ureteric bud (UB), leading to formation of its daughter collecting ducts, calyces, pelvis and ureters. Even subtle defects in the efficiency and/or accuracy of this process have profound effects on the ultimate development of the kidney and result in congenital abnormalities of the kidney and urinary tract. This review summarizes current knowledge regarding a number of genes known to regulate UB development and emphasizes an emerging role for the renin-angiotensin system (RAS) in renal branching morphogenesis. Mutations in the genes encoding components of the RAS in mice cause renal papillary hypoplasia, hydronephrosis, and urinary concentrating defect. These findings imply that UB-derived epithelia are targets for angiotensin (ANG) II actions during metanephric kidney development. Here, it is proposed that papillary hypoplasia in RAS-deficient mice is secondary to an intrinsic defect in the development of the renal medulla. This hypothesis is based on the following observations: a) UB and surrounding stroma express angiotensinogen (AGT) and ANG II AT1 receptors in vivo; b) ANG II stimulates UB cell process extension, branching and cord formation in collagen gel cultures in vitro; and c) AT1 blockade inhibits ANG II-induced UB cell branching. It is further postulated that ANG II is a novel stroma-derived factor involved in stroma/UB cross-talk which regulates UB branching morphogenesis.     

Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney

Semaphorins, originally identified as axon guidance molecules, have also been implicated in angiogenesis, function of the immune system and cancerous growth. Here we show that deletion of Plexin B2 (Plxnb2), a semaphorin receptor that is expressed both in the pretubular aggregates and the ureteric epithelium in the developing kidney, results in renal hypoplasia and occasional double ureters. The rate of cell proliferation in the ureteric epithelium and consequently the number of ureteric tips are reduced in the kidneys lacking Plexin B2 (Plxnb2-/-). Semaphorin 4C, a ligand for Plexin B2, stimulates branching of the ureteric epithelium in wild type and Plxnb2+/- kidney explants, but not in Plxnb2-/- explants. As shown by co-immunoprecipitation Plexin B2 interacts with the Ret receptor tyrosine kinase, the receptor of Glial-cell-line-derived neurotrophic factor (Gdnf), in embryonic kidneys. Isolated Plxnb2-/- ureteric buds fail to respond to Gdnf by branching, but this response is rescued by Fibroblast growth factor 7 and Follistatin as well as by the metanephric mesenchyme. The differentiation of the nephrogenic mesenchyme, its morphology and the rate of apoptosis in the Plxnb2-/- kidneys are normal. Plexin B2 is co-expressed with Plexin B1 (Plxnb1) in the kidney. The double homozygous Plxnb1-Plxnb2-deficient mice show high embryonic lethality prior to onset of nephrogenesis. The only double homozygous embryo surviving to E12 showed hypoplastic kidneys with ureteric branches and differentiating mesenchyme. Taken together, our results show that Sema4C-Plexin B2 signalling regulates ureteric branching, possibly through modulation of Gdnf signalling by interaction with Ret, and suggest non-redundant roles for Plexin B1 and Plexin B2 in kidney development.     

Increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the alpha/beta hydrolase domain containing 2 gene

Multiple steps, including the migration of vascular smooth muscle cells (SMCs), are involved in the pathogenesis of atherosclerosis. To discover genes which are involved in these steps, we screened mutant mouse lines established by the exchangeable gene trap method utilizing X-gal staining during their embryonic development. One of these lines showed strong reporter gene expression in the vitelline vessels of yolk sacs at embryonic day (E) 12.5. The trap vector was inserted into the fifth intron of alpha/beta hydrolase domain containing 2 (Abhd2) gene which was shown to be expressed in vascular and non-vascular SMCs of adult mice. Although homozygous mutant mice were apparently normal, enhanced SMC migration in the explants SMCs culture and marked intimal hyperplasia after cuff placement were observed in homozygous mice in comparison with wild-type mice. Our results show that Abhd2 is involved in SMC migration and neointimal thickening on vascular SMCs.     

BMP signaling is required for septation of the outflow tract of the mammalian heart

Bone morphogenetic proteins (BMPs) constitute a family of approximately 20 growth factors involved in a tremendous variety of embryonic inductive processes. BMPs elicit dose-dependent effects on patterning during gastrulation and gradients of BMP activity are thought to be established through regulation of the relative concentrations of BMP receptors, ligands and antagonists. We tested whether later developmental events also are sensitive to reduced levels of BMP signaling. We engineered a knockout mouse that expresses a BMP type II receptor that lacks half of the ligand-binding domain. This altered receptor is expressed at levels comparable with the wild-type allele, but has reduced signaling capability. Unlike Bmpr2-null mice, mice homozygous for this hypomorphic receptor undergo normal gastrulation, providing genetic evidence of the dose-dependent effects of BMPs during mammalian development. Mutants, however, die at midgestation with cardiovascular and skeletal defects, demonstrating that the development of these tissues requires wild-type levels of BMP signaling. The most striking defects occur in the outflow tract of the heart, with absence of septation of the conotruncus below the valve level and interrupted aortic arch, a phenotype known in humans as persistent truncus arteriosus (type A4). In addition, semilunar valves do not form in mutants, while the atrioventricular valves appear unaffected. Abnormal septation of the heart and valve anomalies are the most frequent forms of congenital cardiac defects in humans; however, most mouse models display broad defects throughout cardiac tissues. The more restricted spectrum of cardiac anomalies in Bmpr2(deltaE2) mutants makes this strain a key murine model to understand the embryonic defects of persistent truncus arteriosus and impaired semilunar valve formation in humans.     

Regulation of oligodendrocyte progenitor cell maturation by PPARδ: effects on bone morphogenetic proteins

In EAE (experimental autoimmune encephalomyelitis), agonists of PPARs (peroxisome proliferator-activated receptors) provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte) maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells), and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins). We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day), GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.