Ca2+ regulation in detrusor smooth muscle from ovine fetal bladder after in utero bladder outflow obstruction.

PURPOSE: We characterized intracellular Ca(2+) regulation in fetal bladders following outflow obstruction by examining the Ca(2+) response to agonists in smooth muscle cells. MATERIALS AND METHODS: Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery. Intracellular Ca(2+) in single smooth muscle cells isolated from the bladder wall was measured with epifluorescence microscopy using fura-2(AM) during exposure to agonists, such as carbachol and adenosine triphosphate, and to other activators, such as caffeine and KCl. RESULTS: Detrusor smooth muscle cells from obstructed bladders had resting intracellular Ca(2+) similar to that in sham operated controls. The maximal response to carbachol was decreased following obstruction (p <0.05). Construction of dose-response curves also demonstrated higher EC(50) (p <0.05). However, these changes were not mirrored by caffeine evoked Ca(2+) release, which was not significantly different between the obstruction group and sham operated controls. Kinetic analysis of carbachol transients further revealed an attenuated maximal rate of increase in obstructed bladders (p <0.01). The magnitude of intracellular Ca(2+) to purinergic neurotransmitter adenosine triphosphate was also found to be smaller in cells from obstructed bladders (p <0.05), although transmembrane influx by high K depolarization was not significantly affected. CONCLUSIONS: Muscarinic and purinergic pathways were down-regulated in fetal detrusor muscle following outflow obstruction. These major functional receptors appeared to be more susceptible to obstruction than other Ca(2+) regulators. Their impairment may contribute to the compromised contractile function seen in in utero bladder outflow obstruction.

Uroplakins: new molecular players in the biology of urinary tract malformations.

The uroplakins (UPs) are a family of proteins which associate with each other and form plaques on the apical surface of the urothelium. These plaques contribute to a permeability barrier, preventing the influx of urine from the urinary tract lumen. Urinary tract malformations associated with human and mouse UP mutations, the human fetal expression patterns of UPs and experiments in Xenopus oocytes are collectively revealing new functions for the UPs, forcing us to view these proteins in a new light. Rather than simply being products of the urothelial differentiation program, they may be a group of proteins central to the process of urinary tract differentiation itself.

Experimental short-term fetal bladder outflow obstruction: I. Morphology and cell biology associated with urinary flow impairment.

PURPOSE: In fetal sheep, combined urethral and urachal obstruction initiated at 75 days' gestation and maintained for 30 days led to dysmorphic bladders, similar to those found in humans with prune belly syndrome, and uniformly disrupted kidney development. We aimed to create a less severe model of fetal bladder outlet obstruction, more closely resembling infants with posterior urethral valves, and additionally to further our understanding on the role of the urachus. We hypothesized that milder morphological renal tract changes would occur after shorter term experimental obstruction. MATERIALS AND METHODS: Male fetal lambs were assigned to urachal and urethral ligation, urachal ligation only or sham operations. Analyses were performed after 9 days. RESULTS: Concurrent urachal and urethral obstruction resulted in increased bladder weight, and protein and DNA content. Detrusor smooth muscle was well maintained, as assessed by light and electron microscopy, although urothelia showed basal apoptosis. Bladder obstruction led to hydronephrosis but failed to produce significant perturbations in urine osmolality. The nephrogenic cortex was either well preserved or was replaced by glomerular cysts; the latter group tended to have heavier bladders. Urachal obstruction alone produced similar changes suggesting that the male sheep fetal urethra is a high-resistance conduit in mid-gestation. CONCLUSIONS: Concurrent urachal and urethral obstruction, or urachal obstruction alone, initiated in mid-gestation and maintained for 9 days leads to bladder overgrowth but preserved renal tubular function. In future, it will be interesting to determine whether bladder decompression around this stage leads to reversal of bladder overgrowth and/or ameliorates severe renal tract damage described after longer term fetal bladder outflow obstruction.

Experimental short-term partial fetal bladder outflow obstruction: II. Compliance and contractility associated with urinary flow impairment.

PURPOSE: Posterior urethral valves (PUV) is the commonest cause of congenital bladder outlet obstruction. Despite valve ablation in the neonatal period, up to 70% of patients develop renal failure by their teenage years, and progressive bladder dysfunction. This study forms part of a continuing project examining the relationship between severity and duration of obstruction and urinary tract dysfunction. Here is the assessed result of short-term (9-day) obstruction. MATERIALS AND METHODS: Fourteen male fetal lambs at 75 days' gestation were assigned to one of three groups: urachal ligation, urachal ligation with partial urethral obstruction, sham-operated controls. Pregnancy proceeded for 9 days. At autopsy, filling cystometry was performed with the urinary tract in situ and the bladder harvested for nerve counts using PGP 9.5 immunohistochemistry, or in vitro measurement of contractile function. RESULTS: Obstruction was associated with an increase in bladder:fetal weight ratio. Compliance was variable in the obstructed bladders, but the calculated wall stress per unit strain was either similar or less than controls. Nerve-mediated or agonist-induced contraction magnitude in tissue from obstructed bladders and nerve counts did not differ from controls. CONCLUSIONS: Nine days of outflow obstruction at mid-gestation generated a bladder of increased weight but without evidence of contractile failure. An increase in bladder compliance as a function of bladder growth was observed even at this stage, and represents one of the initial responses to outflow tract obstruction.

Neurotransmission and viscoelasticity in the ovine fetal bladder after in utero bladder outflow obstruction.

Fetal bladder outflow obstruction, predominantly caused by posterior urethral valves, results in significant urinary tract pathology; these lesions are the commonest cause of end-stage renal failure in children, and up to 50% continue to suffer from persistent postnatal bladder dysfunction. To investigate the physiological development of the fetal bladder and the response to urinary flow impairment, we performed partial urethral obstruction and complete urachal ligation in the midgestation fetal sheep for 30 days. By electrical and pharmacological stimulation of bladder strips, we found that muscarinic, purinergic, and nitrergic mechanisms exist in the developing fetal bladder at this gestation. After bladder outflow obstruction, the fetal bladder became hypocontractile, producing less force after nerve-mediated and muscarinic stimulation with suggested denervation, and also exhibited greater atropine resistance. Furthermore, fetal bladder urothelium exerted a negative inotropic effect, partly nitric oxide mediated, that was not present after obstruction. Increased compliance, reduced elasticity, and viscoelasticity were observed in the obstructed fetal bladder, but the proportion of work performed by the elastic component (a physical parameter of extracellular matrix) remained the same. In addition to denervation, hypocontractility may result from a reduction in the elastic modulus that may prevent any extramuscular components from sustaining force produced by detrusor smooth muscle.

Extracellular matrix protein expression during mouse detrusor development.

BACKGROUND/PURPOSE: Extracellular matrix proteins are implicated in regulating cell proliferation and differentiation. The authors systematically analysed the expression of elastin; collagen types I, III, IV; laminin; and fibronectin during mouse detrusor muscle development, a period during which downregulation of detrusor proliferation and increasing smooth muscle differentiation is known to occur. METHODS: Embryonic days 14 (E14) and 18 (E18), and postnatal day 1 (D1) and week 6 (6wk) were examined, a period spanning the inception of the bladder to postnatal maturity. Immunohistochemistry of whole bladders was used to immunolocalise protein expression, and Western blot of dissected detrusor layers was used to semiquantify soluble protein expression. RESULTS: All proteins were detected at all 4 stages. Statistically significant increases were documented for elastin (E14 to 6wk), collagen type I (E18 to 6wk), collagen type III (D1 to 6wk) and laminin (E14 to 6wk). Fibronectin levels were relatively high up to D1, after which levels declined significantly. Collagen type IV levels decreased significantly (E18 to 6wk). CONCLUSIONS: The authors postulate that changing levels of laminin and fibronectin have opposing effects on the transition from proliferating primitive mesenchymal cells to differentiated detrusor muscle. Furthermore, changes in collagen type III and elastin may be important for bladder compliance.

Smooth muscle differentiation and cell turnover in mouse detrusor development.

PURPOSE: We systematically analyzed detrusor muscle differentiation in normal mice with a focus on cell turnover (proliferation and apoptosis) as well as on expression of the muscle specific proteins alpha-smooth muscle actin and desmin. MATERIALS AND METHODS: The stages examined were embryonic days 14 and 18, and postnatal day 1 and week 6, representing a period spanning organ inception to postnatal maturity. Alpha-smooth muscle actin, desmin and proliferating cell nuclear antigen were assessed by immunohistochemical testing of whole bladders and Western blot analysis of dissected detrusor layers. Apoptosis was detected in tissue sections by end-labeling. RESULTS: Alpha-smooth muscle actin was expressed by the detrusor layer throughout maturation with levels significantly increasing from embryonic days 14 to 18 and cytoplasmic staining gaining in uniformity postnatally. Desmin expression in the detrusor was insignificant at embryonic day 14 but increased progressively thereafter. Proliferating cell nuclear antigen expression in the detrusor was highest at organ inception and fell stepwise at each developmental stage with low levels postnatally. Apoptosis in the detrusor was only detected at embryonic day 14. CONCLUSIONS: These results demonstrate that morphological growth of the mouse detrusor muscle is accompanied by complex serial changes in the expression of muscle specific proteins and in cell turnover. Strikingly, detrusor muscle cell differentiation and proliferation are inversely related. These detailed studies may serve as a comparison for future experiments involving aberrant mouse bladder development.

Embryonic gut anomalies in a mouse model of retinoic Acid-induced caudal regression syndrome: delayed gut looping, rudimentary cecum, and anorectal anomalies.

Vitamin A and its derivatives such as retinoic acid (RA) are important signaling molecules for morphogenesis of vertebrate embryos. Little is known, however, about morphogenetic factors controlling the development of the gastrointestinal tract and RA is likely to be involved. In the mouse, teratogenic doses of RA cause truncation of the embryonic caudal body axis that parallel the caudal regression syndrome as described in humans. These changes are often associated with anomalies of the lower digestive tract. Overlapping spatiotemporal expression of retinoic acid receptor-beta (RAR beta) and cellular retinol-binding protein I, CRBPI, with Hoxb5 and c-ret in the gut mesoderm imply possible cooperation required for proper neuromuscular development. To determine susceptibility and responsiveness of the developing gut and its neuromusculature to exogenous retinoids we used a mouse model of RA-induced caudal regression syndrome. The results showed that stage-specific RA treatment both in vivo and in vitro affected gut looping/rotation morphogenesis and growth of asymmetrical structures such as the cecum together with delayed differentiation of the gut mesoderm and colonization of the postcecal gut by neural crest-derived enteric neuronal precursors. These observations demonstrate that RA has a direct effect on gut morphogenesis and innervation.

Congenital obstructive uropathy: its origin and contribution to end-stage renal disease in children.

Most children with end-stage renal failure are born with abnormal kidneys, with fetal obstructive uropathy accounting for about half these cases. Posterior urethral valves (PUV) is overwhelmingly the most common, specific diagnosis, and is confined to boys. In general, the condition is sporadic, although familial cases have been reported, perhaps suggesting that genetic factors play a role in pathogenesis. PUV can present antenatally, detected on routine fetal ultrasonography, or postnatally, when it is associated with renal failure and bladder dysfunction. Advances in postnatal surgery and medical management have reduced the mortality in PUV, but it is uncertain whether antenatal diagnosis or treatment (eg, with vesicoamniotic shunts) improves long-term renal outcome. Other disorders associated with human fetal obstructive uropathy are urethral atresia, the prune belly syndrome, and multicystic dysplastic kidney. The nephropathy associated with fetal obstructive uropathy is renal dysplasia and comprises undifferentiated and metaplastic tissues. Several studies in animals and in humans suggest that fetal urine flow impairment causes deregulation of renal precursor cell turnover and expression of growth factor/survival and transcription factor genes. Furthermore, some of these morphologic and molecular changes can be ameliorated experimentally by fetal surgical decompression or administration of specific growth factors.

Expression and potential role of angiopoietins and Tie-2 in early development of the mouse metanephros.

Angiopoietins (Ang) are secreted factors which bind the Tie-2 receptor and modulate endothelial growth. This signalling system is known to be expressed in later stages of maturation of the mouse metanephros, the adult kidney precursor. In this study, by using reverse transcription polymerase chain reaction and Northern and Western blotting, we demonstrated that Ang-1, Ang-2, and Tie-2 were expressed during early metanephrogenesis when interstitial and glomerular capillaries begin to form. By using immunohistochemistry, embryonic kidney capillaries in the interstitium and glomeruli expressed Tie-2 at a later stage of differentiation compared with vascular endothelial growth factor receptor-2 and platelet-endothelial cell adhesion molecule. Addition of 200 ng/ml Ang-1 to explanted embryonic day (E) 12.5 metanephroi increased the proportion of vascular glomeruli that formed during 1 week in culture. These results are consistent with the hypotheses that Tie-2 has a role in vascular growth in the early stages of mammalian nephrogenesis and that Tie-2 activation may maintain the integrity of recently formed interstitial and glomerular vessels.

Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

Deregulation of renal transforming growth factor-beta1 after experimental short-term ureteric obstruction in fetal sheep.

Renal malformations are the commonest cause of chronic renal failure in children and they are often associated with urinary tract abnormalities that impair fetal urine flow. Up-regulation of transforming growth factor-beta1 (TGF-beta1) occurs after experimental postnatal urinary tract obstruction and we recently reported increased levels of TGF-beta1 in human renal malformations (Yang SP et al, Am J Pathol 2000, 157:1633-1647). These findings led us to propose that obstruction-induced stretch of developing renal epithelia causes up-regulation of TGF-beta1, which then perturbs renal development. In this study, therefore, we examined expression of components of the TGF-beta1 signaling axis in a previously characterized ovine model of fetal short-term urine flow impairment in which complete unilateral ureteric obstruction was induced at 90 days when a few layers of glomeruli had formed. Up-regulation of TGF-beta1 mRNA and protein was observed in obstructed kidneys, compared to sham-operated control organs, after only 10 days. Increased levels of TGF-beta1 receptors I (TGF-betaR1) and II (TGF-betaR2) were also detected on Western blot, and the cytokine and TGF-betaR1 co-localized in disrupted epithelia on immunohistochemistry. De novo expression of alpha-smooth muscle actin, a structural protein up-regulated during TGF-beta1-induced phenotypic switching between human renal dysplastic epithelial and mesenchymal lineages in vitro, was also observed in these aberrant epithelia. These findings implicate increased TGF-beta1 signaling in the early biological changes generated by fetal urinary tract obstruction.

Multicystic dysplastic kidney and Kallmann's syndrome: a new association?

BACKGROUND: Kallmann's syndrome is characterized by anosmia and hypogonadotrophic hypogonadism. Radiographic studies of teenagers and older subjects with the X-linked form of the syndrome have shown that up to 40% have an absent kidney unilaterally. Although this has been attributed to renal "agenesis", a condition in which the kidney fails to form, little is known about the appearance of the developing urinary tract either pre- or post-natally in individuals with Kallmann's syndrome. METHODS: We describe two brothers who had features of Kallmann's syndrome, most probably of the X-linked variety, who both had a major urinary-tract malformation detected before birth. RESULTS: The brothers were found to have unilateral multicystic dysplastic kidneys on routine antenatal ultrasound scanning and both underwent surgical nephrectomy of these organs post-natally. Immunohistochemical studies on the younger sibling revealed hyperproliferative dysplastic kidney tubules which overexpressed PAX2, a potentially oncogenic transcription factor, and BCL2, a cell-survival factor, surrounded by metaplastic, alpha smooth-muscle actin-positive stroma: similar patterns have been observed in patients with non-syndromic multicystic dysplastic kidneys. CONCLUSIONS: Our results describe a new type of urinary-tract malformation associated with Kallmann's syndrome. However, since multicystic kidneys tend to involute, only when more Kallmann's syndrome patients are screened in utero or in early childhood using structural renal scans, will it be possible to establish whether multicystic kidney disease is a bona-fide part of the syndrome.

Medical versus surgical treatment in children with severe bilateral vesicoureteric reflux and bilateral nephropathy: a randomised trial.

BACKGROUND: Nephropathy associated with vesicoureteric reflux (VUR) and urinary tract infection can result in end-stage renal failure, hypertension, or both. Whether long-term VUR contributes to these outcomes is unknown. We compared, in a randomised trial, medical with surgical management of children with bilateral severe VUR and bilateral nephropathy. METHODS: We stratified by age and glomerular filtration rate (GFR) 25 boys and 27 girls aged 1-12 years and randomly assigned them to medical or surgical management. At enrolment and 4 years' follow-up we estimated GFR from the plasma clearance of 51Cr-labelled edetic acid (EDTA), and did intravenous urography. We also did a metastable 99mTc-labelled dimercaptosuccinic acid (DMSA) assay and contrast cystography. The change in GFR at 4 years, expressed as a percentage change between enrolment and 4 years, was available for 26 of 27 patients in the medical and 24 of 25 in the surgical group. We assessed GFR in 48 patients 10 years after enrolment. FINDINGS: Mean GFR at enrolment was 72.4 mL/min per 1.73 m(2) (SD 24.1) in the medical and 71.7 mL/min per 1.73 m(2) (22.6) in the surgical group. The mean percentage change in GFR at 4 years was 2.4% (SE 4.5) versus 4.7% (5.0) in the medical and surgical groups, respectively. The difference in change in GFR at 4 years between the two groups was not significant (7.1%, 95% CI 6.4% to 20.6%). INTERPRETATION: Our data do not lend support to the view that the outcome for renal function is improved by surgical correction of VUR in children with bilateral disease.

Angiopoietin growth factors and Tie receptor tyrosine kinases in renal vascular development.

Angiopoietin-1 (Ang-1) is a secreted growth factor which binds to and activates the Tie-2 receptor tyrosine kinase. The factor enhances endothelial cell survival and capillary morphogenesis, and also limits capillary permeability. Ang-2 binds the same receptor but fails to activate it: hence, it is a natural inhibitor of Ang-1. Ang-2 destabilises capillary integrity, facilitating sprouting when ambient vascular endothelial growth factor (VEGF) levels are high, but causing vessel regression when VEGF levels are low. Tie-1 is a Tie-2 homologue but its ligands are unknown. Angiopoietin and Tie genes are expressed in the mammalian metanephros, the precursor of the adult kidney, where they may play a role in endothelial precursor growth. Tie-1-expressing cells can be detected in the metanephros when it first forms and, based on transplantation experiments, these precursors contribute to the generation of glomerular capillaries. During glomerular maturation, podocyte-derived Ang-1 and mesangial-cell-derived Ang-2 may affect growth of nascent capillaries. After birth, vasa rectae acquire their mature configuration and Ang-2 expressed by descending limbs of loops of Henle would be well placed to affect the growth of this medullary microcirculation. Finally, preliminary data implicate angiopoietins in deregulated vessel growth in Wilms' kidney tumours and in vascular remodelling after nephrotoxicity.

Identification of the gene for oral-facial-digital type I syndrome.

Oral-facial-digital type 1 syndrome (OFD1 [MIM 311200]) is transmitted as an X-linked dominant condition with lethality in males and is characterized by malformations of the face, oral cavity, and digits, and by a highly variable expressivity even within the same family. Malformation of the brain and polycystic kidneys are commonly associated with this disorder. The locus for OFD1 was mapped by linkage analysis to a 12-Mb interval, flanked by markers DXS85 and DXS7105 in the Xp22 region. To identify the gene responsible for this syndrome, we analyzed several transcripts mapping to the region and found mutations in OFD1 (formerly named "Cxorf5/71-7a"), encoding a protein containing coiled-coil alpha-helical domains. Seven patients with OFD1, including three with familial and four with sporadic cases, were analyzed. Analysis of the familial cases revealed a missense mutation, a 19-bp deletion, and a single base-pair deletion leading to a frameshift. In the sporadic cases, we found a missense (de novo), a nonsense, a splice, and a frameshift mutation. RNA in situ studies on mouse embryo tissue sections show that Ofd1 is developmentally regulated and is expressed in all tissues affected in OFD1 syndrome. The involvement of OFD1 in oral-facial-digital type I syndrome demonstrates an important role of this gene in human development.

Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease.

Familial glomerulocystic kidney disease (GCKD) is a dominantly inherited condition characterized by glomerular cysts and variable renal size and function; the molecular genetic etiology is unknown. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with early-onset diabetes and nondiabetic renal disease-particularly renal cystic disease. We investigated a possible role for the HNF-1beta gene in four unrelated GCKD families and identified mutations in two families: a nonsense mutation in exon 1 (E101X) and a frameshift mutation in exon 2 (P159fsdelT). The family members with HNF-1beta gene mutations had hypoplastic GCKD and early-onset diabetes or impaired glucose tolerance. We conclude that there is genetic heterogeneity in familial GCKD and that the hypoplastic subtype is a part of the clinical spectrum of the renal cysts and diabetes syndrome that is associated with HNF-1beta mutations.