Urachal Ligation Research Articles

Urothelial Vascular Endothelial Growth Factor-a Expression in Fetal Bladder Outflow Obstruction

PURPOSE Vascular endothelial growth factor-A (VEGF-A) directs blood vessel formation in development and disease. Recently, it was demonstrated that VEGF-A enhanced embryonic bladder growth in organ culture, both in the urothelial and detrusor smooht muscle compartments. Therefore, we hypothesised that VEGF-A may play a role in diseases where bladder overgrowth occurs such as bladder outflow obstruction. MATERIAL AND METHODS VEGF-A expression was examined in the bladders of fetal sheep that had undergone short- (9 days) or long-term (30 days) bladder obstruction by combined urethral obstruction and urachal ligation at 75 days gestation. Short-term obstructuction leads to overgrowth of the whole bladder, resembling infants born with posterior urethral valves. When obstruction is maintained for 30 days, massively-dilated, thin walled bladders are poorly contractile, a phenotype resembling either the demcompensated bladder seen in some teenagers with posterior urthral valves or the prune belly syndrome. RESULTS VEGF-A was immunolocalised to the urothelium of the bladder in all experimental groups. In short-term obstruction, VEGF-A expression was upregulated versus time-matched controls and this group tended to have urothelium with multiple layers of nuclei. Conversely, animals obstructed for 30 days had patchy downregulation of VEGF-A expression in the urothelium which had become considerably thinner with few layers of nuclei. CONCLUSIONS VEGF-A is expressed in the fetal urothelium. There appears to be a biphasic response after bladder outflow obstruction and it is possible that increased VEGF-A plays a role in the actual growth response in this model.

Ca 2+ Regulation in Detrusor Smooth Muscle From Ovine Fetal Bladder After In Utero Bladder Outflow Obstruction

We characterized intracellular Ca(2+) regulation in fetal bladders following outflow obstruction by examining the Ca(2+) response to agonists in smooth muscle cells. 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. 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. 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.

Early bladder wall changes after creation of obstructive uropathy in the fetal lamb

Vesico-amniotic shunting of obstructive uropathy in fetal lambs produced a thick-walled, poorly compliant bladder. We report the early histological changes in the obstructed bladder wall. We created an obstructive uropathy in fetal lambs at 60 days gestation by ligating the urethra and urachus. Vesicostomy or vesico-amniotic shunt tube insertion and biopsy of the bladder wall were performed 21 days later. The fetuses were delivered at term (145 days) and the kidneys and bladder sampled for histology. Colloidal iron (Col Fe), and alpha-smooth muscle actin (alpha-SMA) immunohistochemical stains were used for these samples. Seventeen fetuses were shunted with 15 biopsies taken at that time. Six (shunt failure or missed urachal ligation) were excluded. All biopsies taken at shunting had positive Col Fe and alpha-SMA. Term lambs had mild multicystic dysplastic kidney (MCDK) in five, severe MCDK in two, and hydronephrosis in four. All bladders had small volume and were severely fibrotic. Fetal shunt operations 3 weeks after the creation of obstructive uropathy provided partial preservation of renal histology but did not preserve normal bladder histology. We suggest that the high hyaluronic acid synthesis activity or hyperplasia of the myofibroblasts in the dilated fetal bladder wall at the time of shunting results in irreversible damage to the developing bladder muscle and fibrosis.

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.

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.

Effects of in utero bladder outflow obstruction on fetal sheep detrusor contractility, compliance and innervation.

Congenital bladder outflow obstruction caused by posterior urethral valves is a common cause of end stage renal failure in boys. We hypothesized that fetal bladder outflow obstruction perturbs detrusor contractility and innervation and bladder storage volume-pressure relationships. 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, when urinary tract dilatation, enlarged bladders and histologically abnormal kidneys were documented. Isolated strips of bladder detrusor from sham operated and obstructed fetuses were subjected to electrical field stimulation, carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Whole bladder storage characteristics were determined by filling cystometry and bladder innervation was investigated by immunohistochemistry and Western blot. Tension-frequency contractility studies showed that obstructed fetal bladder strips were significantly hypocontractile versus sham operated controls in response to electrical field stimulation and the specific agonists carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Hypocontractility was greater with nerve mediated stimulation than with carbachol, suggesting relative denervation. Reduced innervation was confirmed by S100 and protein gene product 9.5 immunohistochemistry and by measuring a significant reduction in protein gene product 9.5 protein expression using Western blot. Filling cystometry showed that obstructed fetal bladders appeared more compliant (Delta V/Delta P, where Delta V is the change in volume and Delta P is the change in pressure) with larger capacity, more flaccidity and yet retained stress relaxation. In response to severe experimental fetal bladder outflow obstruction the bladder becomes large and hypocontractile, and has aberrant innervation.

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.

Effects of In Utero Bladder Outflow Obstruction on Fetal Sheep Detrusor Contractility, Compliance and Innervation

Congenital bladder outflow obstruction caused by posterior urethral valves is a common cause of end stage renal failure in boys. We hypothesized that fetal bladder outflow obstruction perturbs detrusor contractility and innervation and bladder storage volume-pressure relationships. 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, when urinary tract dilatation, enlarged bladders and histologically abnormal kidneys were documented. Isolated strips of bladder detrusor from sham operated and obstructed fetuses were subjected to electrical field stimulation, carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Whole bladder storage characteristics were determined by filling cystometry and bladder innervation was investigated by immunohistochemistry and Western blot. Tension-frequency contractility studies showed that obstructed fetal bladder strips were significantly hypocontractile versus sham operated controls in response to electrical field stimulation and the specific agonists carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Hypocontractility was greater with nerve mediated stimulation than with carbachol, suggesting relative denervation. Reduced innervation was confirmed by S100 and protein gene product 9.5 immunohistochemistry and by measuring a significant reduction in protein gene product 9.5 protein expression using Western blot. Filling cystometry showed that obstructed fetal bladders appeared more compliant (Delta V/Delta P, where Delta V is the change in volume and Delta P is the change in pressure) with larger capacity, more flaccidity and yet retained stress relaxation. In response to severe experimental fetal bladder outflow obstruction the bladder becomes large and hypocontractile, and has aberrant innervation.

Different phenotypes of dysplastic kidney in obstructive uropathy in fetal lambs

Purpose: The cause of cyst production in renal dysplasia is uncertain. The authors hypothesized that different patterns of renal dysplasia result from variations in the timing and site of the urinary tract obstruction. Methods: The authors operated on fetal lambs at 50 and 60 days' gestation. Male lambs underwent urethral and urachal ligation and female lambs unilateral ureteric ligation. They were delivered by cesarean section at 145 days' gestation and killed. Results: Of 12 lambs operated on at 50 days' gestation, 4 survived. Of 26 lambs operated on at 60 days, 21 survived. The authors identified 3 types of dysplastic kidneys. Type A, fibrotic kidneys (2.2 g) with no cysts and interstitial fibrosis. There were reduced numbers of proximal tubules, but distal tubules and collecting ducts persisted. (50-day obstruction, n = 5 kidneys); type B, Sponge-like kidneys (37g): these had large cysts with minimal interstitial fibrosis. (87% of 60-day uretheral and urachal ligation model n = 12 kidneys); Type C, Small kidneys (4.8 g) with no large cysts (60-day Ureteric ligation model n = 7 kidneys). Conclusion: The authors produced 3 different types of renal dysplasia by creating urinary tract obstruction at different sites and gestational ages. J Pediatr Surg 36:1698-1703. Copyright © 2001 by W.B. Saunders Company.

Early fetal obstructive uropathy produces Potter's syndrome in the lamb

Background: If creating an obstructive uropathy early in glomerulogenesis produces MCDK (Multicystic Dysplastic Kidney), then a very early obstruction may produce Potter's Syndrome (PS) with oligohydramnios. Methods: Fetal lambs at 50 days' gestation underwent urethral and urachal ligation using fine SILASTIC® (Dow Corning, Midland, MI) tubing and were delivered by cesarean section at 145 days' gestation. At the time of death, kidney weight, length, and lung volumes were measured. These samples were examined histologically. Urinary sodium, chloride, potassium, and osmolarity also were measured. These were compared with normal-term fetuses. Results: One ewe miscarried. Two of 3 of 50-day obstructive uropathy lambs survived. The 2 survivors had dysplastic kidneys. One with large gastroschisis did not have PS but the other had renal, pulmonary, and chest wall hypoplasia. Both male lambs had undescended testes with a large bladder. Kidney weights were 2 g in the PS lamb and 16 g in controls. Lung volume was 84 mL in the PS lamb and 340 mL in controls. The lamb's face was compressed and the fetus was hydropic. Urine sodium, potassium, and osmolarity levels were higher than that of controls. Conclusions: This is the first successful model ligating the penile urethra and urachus in a 50-day lamb. The authors' previous 60-day model did not have PS, but an earlier obstructive uropathy caused MCDK with PS. J Pediatr Surg 35:1549-1553. Copyright © 2000 by W.B. Saunders Company.

EXPERIMENTAL VESICOURETERAL REFLUX IN THE FETUS DEPENDS ON BLADDER FUNCTION AND CAUSES RENAL FIBROSIS

Purpose Prenatal diagnosis allows the early detection of vesicoureteral reflux in an increasing number of newborns, some of whom are born with impaired kidney function. This situation challenges our current understanding of the pathophysiology, natural history and, therefore, treatment of reflux. We created a fetal sheep model of vesicoureteral reflux to study the mechanisms of fetal reflux nephropathy. Materials and Methods Vesicoureteral reflux was induced in fetal sheep at 95 days of gestation (term 140 days) by open bladder incision of the intravesical ureteral tunnel. All animals underwent urachal ligation and in female subjects mild bladder outlet obstruction was created with a gold ring. Results At term reflux was detected in 18 of 28 renal units by filling cystography. Refluxing kidneys were hydronephrotic and larger than normal. At term mean kidney weight was 21.1 gm. (range 12.2 to 35.0) in male subjects with reflux compared to 8.5 gm. (range 6.5 to 11.3) in normal male subjects (p <0.001) and 11.5 gm. (range 8.5 to 15.8) in male subjects with urachal ligation only (p = 0.035). In female subjects there was no change in renal weight. Renal histology revealed a thin, structurally normal cortex with small subcortical cysts and a hypoplastic medulla with mesenchymal tissue replacing normal ducts. Total mean renal collagen content was significantly increased to 51.7 mg. (range 35 to 81) in the refluxing kidneys of male animals, while it was 23.8 mg. (range 12.1 to 38.4) in normal male animals (p = 0.03). The fractional excretion of sodium was elevated in refluxing kidneys based on sodium-to-creatinine ratios in bladder urine. Conclusions In a novel model of fetal vesicoureteral reflux we showed that prenatal reflux nephropathy is characterized by altered renal growth regulation, structural maldevelopment without overt dysplasia, excess matrix deposition and impaired excretory function.

Experimental vesicoureteral reflux in the fetus depends on bladder function and causes renal fibrosis.

Prenatal diagnosis allows the early detection of vesicoureteral reflux in an increasing number of newborns, some of whom are born with impaired kidney function. This situation challenges our current understanding of the pathophysiology, natural history and, therefore, treatment of reflux. We created a fetal sheep model of vesicoureteral reflux to study the mechanisms of fetal reflux nephropathy. Vesicoureteral reflux was induced in fetal sheep at 95 days of gestation (term 140 days) by open bladder incision of the intravesical ureteral tunnel. All animals underwent urachal ligation and in female subjects mild bladder outlet obstruction was created with a gold ring. At term reflux was detected in 18 of 28 renal units by filling cystography. Refluxing kidneys were hydronephrotic and larger than normal. At term mean kidney weight was 21.1 gm. (range 12.2 to 35.0) in male subjects with reflux compared to 8.5 gm. (range 6.5 to 11.3) in normal male subjects (p <0.001) and 11.5 gm. (range 8.5 to 15.8) in male subjects with urachal ligation only (p = 0.035). In female subjects there was no change in renal weight. Renal histology revealed a thin, structurally normal cortex with small subcortical cysts and a hypoplastic medulla with mesenchymal tissue replacing normal ducts. Total mean renal collagen content was significantly increased to 51.7 mg. (range 35 to 81) in the refluxing kidneys of male animals, while it was 23.8 mg. (range 12.1 to 38.4) in normal male animals (p = 0.03). The fractional excretion of sodium was elevated in refluxing kidneys based on sodium-to-creatinine ratios in bladder urine. In a novel model of fetal vesicoureteral reflux we showed that prenatal reflux nephropathy is characterized by altered renal growth regulation, structural maldevelopment without overt dysplasia, excess matrix deposition and impaired excretory function.

Creation of experimental urethral obstruction in utero: evaluation of fetal renal function.

Management of obstructive uropathy diagnosed in utero would be markedly enhanced by the availability of a simple, safe and quantitative fetal renal function test to predict postnatal renal function. In order to allow experimental evaluation of such a parameter, we adjusted a model of complete urethral obstruction with urachal ligation in 30 fetal lambs at 79 or 99 days of gestation. The method described allows obstruction in male and female fetuses as early as 79 days of gestation, with an overall high survival rate (control: 12/14; obstructed: 23/29), although lower (6/12) when obstruction is performed early (79 days) during gestation. Consequences of obstruction were examined on the 121st day of gestation. Severe hydronephrosis, ureteral and calyceal dilatation, with or without ascites and pulmonary hypoplasia were observed in all fetuses; creatinine clearance determined in utero was decreased in both groups with obstruction (early and late) vs control group: 1.15 +/- 0.5, NS, and 0.58 +/- 0.4, p < 0.01 vs 1.61 +/- 0.8 ml/min/kg respectively. In both obstructed groups, fetuses with ascites displayed lower plasma creatinine concentration and higher creatinine clearance values when compared to fetuses without ascites. In conclusion, the experimental model of obstructive uropathy described appears efficient and easily reproducible, allowing therefore the evaluation of a predictive parameter of postnatal renal function. Our preliminary results suggest that renal fetal function is more dependent on the degree of obstruction than on the term of its creation.

Correction of congenital hydronephrosis in utero II. Decompression reverses the effects of obstruction on the fetal lung and urinary tract

Urethral obstruction and urachal ligation at 93-107 days gestation produced severe hydronephrosis, hydroureter, megacystis, and urinary ascites as well as significant pulmonary hypoplasia in 17 fetal lambs. Obstructions in 9 fetuses subsequently were relieved in utero by suprapubic cutaneous cystostomy. At birth, all 4 liveborn obstructed lambs had respiratory insufficiency, and only 1 survived. Four others were stillborn. The lungs were significantly hypoplastic by weight and volume (p less than 0.025). All 8 had advanced megacystis, hydroureter, and hydronephrosis but no cystic or dysplastic renal changes. In contrast, 7 liveborn lambs diverted in utero had far less respiratory difficulty and all survived (p = 0.002). Two were stillborn. The lung weight was significantly increased (p less than 0.05). All lambs undergoing in utero decompression showed significant resolution of the severe urinary tract dilatation seen in the obstructed lambs. In utero decompression of the obstructed fetal urinary tract allows the abnormally small lungs to grow and develop and hydronephrosis to resolve.