The embryology of persistent cloaca and urogenital sinus malformations.

Cloacal malformations are characterized by the confluence of the lower urinary tract, the female reproductive tract, and the rectum to create a common channel with a single opening on the perineum. The presence of a cloaca is a normal phase of early human embryological development. Between the 4th and 7th weeks of gestation, the cloaca undergoes subdivision to form the hindgut and urogenital sinus. Failure of this process results in the congenital anomaly termed persistent cloaca (PC). The term urorectal septum malformation sequence (URSMS) is also used to describe this anomaly. The classic description of this process which is still cited in many standard textbooks dates from the 19th century. However, this has been increasingly called into question by the findings of studies using modern scientific methodology. Urogenital sinus anomalies are defined by the confluence of the urethra and vagina to form a common channel of varying length with a single perineal opening. In this condition, the anorectal canal opens separately on the perineum. The presence of a urogenital sinus represents a transient phase of the normal development of the lower genital tract in the female fetus. However, the form of urogenital sinus most commonly encountered in the developed world is a feature of disordered sexual differentiation and does not arise simply from the persistence of the anatomical structure which is a feature of normal fetal development.

Seromuscular grafts for bladder reconstruction: extra-luminal demucosalisation of the bowel.

OBJECTIVE: To develop a robust sterile, fully demucosalized and vascularized seromuscular patch for use as an adjunct to novel bioengineering techniques aimed at augmenting, reconstructing, or replacing the bladder because of endstage disease. To eliminate deep colonic epithelial crypts to prevent the possibility of colonocyte regrowth. To maintain sterility by excluding the possibility of contamination from the bowel contents. METHODS: Pilot studies were performed on euthanized pigs to optimize the technique, with tissue samples examined by immunohistochemistry. In vivo, vascularized seromuscular colonic flaps were created from the bowel exterior in 7 large white hybrid pigs. The dissection was facilitated by placing an inflated Foley catheter within the colonic lumen. The seromuscular ends were approximated with 5/0 Vicryl sutures and excess mucosa intussuscepted within the lumen. Demucosalized flaps were used to augment the bladder by composite cystoplasty and were examined immunohistochemically at 3 months. RESULTS: Pilot studies showed that the technique was successful in creating seromuscular segments with no epithelial remnants. When applied surgically, the seromuscular flaps survived and showed no evidence of colonocyte regrowth at 3 months. CONCLUSION: Extraluminal dissection creates robust seromuscular flaps and prevents both regrowth by colonic epithelial cells and contamination of the tissue by exposure to the bowel contents. This technique should find application in a range of bladder reconstruction techniques, including composite cystoplasty and autoaugmentation.

Regenerative medicine in urology.

The term 'regenerative medicine' encompasses strategies for restoring or renewing tissue or organ function by: (i) in vivo tissue repair by in-growth of host cells into an acellular natural or synthetic biomaterial, (ii) implantation of tissue 'engineered'in vitro by seeding cultured cells into a biomaterial scaffold, and (iii) therapeutic cloning and stem cell-based tissue regeneration. In this article, we review recent developments underpinning the emerging science of regenerative medicine and critically assess where successful implementation of novel regenerative medicine approaches into urology practice might genuinely transform the quality of life of affected individuals. We advocate the need for an evidence-based approach supported by strong science and clinical objectivity.

Transplantation of autologous differentiated urothelium in an experimental model of composite cystoplasty.

BACKGROUND: Enterocystoplasty is associated with serious complications resulting from the chronic interaction between intestinal epithelium and urine. Composite cystoplasty is proposed as a means of overcoming these complications by substituting intestinal epithelium with tissue-engineered autologous urothelium. OBJECTIVE: To develop a robust surgical procedure for composite cystoplasty and to determine if outcome is improved by transplantation of a differentiated urothelium. DESIGN, SETTING, AND PARTICIPANTS: Bladder augmentation with in vitro-generated autologous tissues was performed in 11 female Large-White hybrid pigs in a well-equipped biomedical centre with operating facilities. Participants were a team comprising scientists, urologists, a veterinary surgeon, and a histopathologist. MEASUREMENTS: Urothelium harvested by open biopsy was expanded in culture and used to develop sheets of nondifferentiated or differentiated urothelium. The sheets were transplanted onto a vascularised, de-epithelialised, seromuscular colonic segment at the time of bladder augmentation. After removal of catheters and balloon at two weeks, voiding behaviour was monitored and animals were sacrificed at 3 months for immunohistology. RESULTS AND LIMITATIONS: Eleven pigs underwent augmentation, but four were lost to complications. Voiding behaviour was normal in the remainder. At autopsy, reconstructed bladders were healthy, lined by confluent urothelium, and showed no fibrosis, mucus, calculi, or colonic regrowth. Urothelial morphology was transitional with variable columnar attributes consistent between native and augmented segments. Bladders reconstructed with differentiated cell sheets had fewer lymphocytes infiltrating the lamina propria, indicating more effective urinary barrier function. CONCLUSIONS: The study endorses the potential for composite cystoplasty by (1) successfully developing reliable techniques for transplanting urothelium onto a prepared, vascularised, smooth muscle segment and (2) creating a functional urothelium-lined augmentation to overcome the complications of conventional enterocystoplasty.

Ureterocalicostomy in children: 12 years experience in a single centre.

OBJECTIVE: • To document the outcome of ureterocalicostomy in children. PATIENTS AND METHODS: • The outcomes of 13 children who had undergone ureterocalicostomy consecutively under the care of two paediatric urologists between 1997 and 2009 were evaluated retrospectively. • Ureterocalicostomy was performed as the primary procedure in four children with horseshoe kidney and four children presenting with gross pelvi-ureteric junction (PUJ) obstruction. • In the remaining five children, it was performed as a secondary procedure for recurrent PUJ obstruction after previous pyeloplasty. • An open approach was employed in 12 patients, whereas, in one patient, it was performed by a laparoscopically-assisted technique. RESULTS: • Mean age at operation was 9.3 years and the mean (range) duration of follow-up was 2.6 (0.3-7.0) years. Twelve children (92%) experienced a good functional outcome following ureterocalicostomy, as defined by reduced dilatation and improved drainage on postoperative ultrasonography and/or isotope imaging. • However one child (8%) developed symptomatic anastomotic obstruction 5 months after primary ureterocalicostomy for obstruction in a horseshoe kidney. Surgical revision was successful, with good drainage, preservation of differential function and relief of symptoms on further follow-up to 3 years. CONCLUSIONS: • Ureterocalicostomy provides a versatile and reliable means of relieving obstruction for a variety of indications, including horseshoe kidney, recurrent PUJ obstruction and gross PUJ obstruction with unfavourable anatomy. • Approximation of ureteric and caliceal urothelium and excision of renal parenchyma in the proximity to the anastomosis are the key steps for securing a satisfactory outcome.

Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro.

BACKGROUND: Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. OBJECTIVE: To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. DESIGN, SETTING, AND PARTICIPANTS: AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. MEASUREMENTS: Qualitative study. RESULTS AND LIMITATIONS: Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. CONCLUSIONS: This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

The long-term outcome of prenatally detected posterior urethral valves: a 10 to 23-year follow-up study.

OBJECTIVE: To document the functional outcome of patients with prenatally detected posterior urethral valves (PUV) in the second decade of life, and to evaluate the possible impact of prenatal diagnosis on the long-term outcome of this condition. PATIENTS AND METHODS: We analysed the functional outcome of 25 patients with prenatally detected PUV born between 1984 and 1996, whose mean (range) age at follow-up was 17.7 (10-23) years. The findings were compared with those in 17 patients (mean age 16.1 years) who had presented clinically to our unit during the same period. The duration of follow-up in both groups was >or=10 years. Late outcomes were also compared with published data for PUV. Outcome measures included; death, incidence of end-stage renal failure (ESRF), age at transplantation and the most the recently available plasma creatinine level in untransplanted patients. We also examined any possible association between functional outcome and early predictors, including nadir plasma creatinine level at <1 year and vesico-ureteric reflux (VUR). RESULTS: Three patients died (12%), two as neonates and one aged 3 years. Of five patients who had been shunted in utero, four died or developed early-onset renal failure. In the 23 prenatally detected patients who survived the neonatal period, four (17%) had a renal transplant at a mean (range) age of 6.5 (3.0-12.0) years. Of 19 patients with prenatally detected PUV who had not been transplanted in the first 12 years of life, only one (5%) developed new-onset ESRF at 10.0-23.4 years whilst 11 (58%) of these patients had normal creatinine values. In the untransplanted patients there was a statistically significant correlation between age and plasma creatinine level, but no correlation between late functional outcome and nadir creatinine in the first year of life, or bilateral VUR. CONCLUSIONS: Prenatal diagnosis had little impact on mortality or ESRF in the first decade of life. This appears to be largely predetermined by renal dysplasia and the severity of intrauterine obstruction. However, the functional outcome of patients with prenatally detected PUV aged 10-23 years was considerably better than published long-term data and the outcome of clinically presenting patients in our study. These findings suggest that the long-term prognosis of PUV of intermediate severity might be improved by prenatal diagnosis.

Generation of a functional, differentiated porcine urothelial tissue in vitro.

BACKGROUND: The primary function of urothelium is to serve as a physical urinary barrier. This function is dependent on features expressed at the molecular level that are acquired during cytodifferentiation. Urothelial cells lose differentiated and functional characteristics when propagated in vitro. OBJECTIVE: To investigate methods of inducing molecular and functional differentiation of normal porcine urothelial (NPU) cells in vitro. DESIGN AND MEASUREMENTS: NPU cells were isolated from normal porcine bladders and propagated in a low-calcium keratinocyte serum-free medium. Effects of 5% fetal bovine serum (FBS) and exogenous calcium were investigated. Molecular differentiation was assessed by immunolabelling for urothelial differentiation-associated proteins (UPIIIa, CK20, ZO-1), and barrier function was assessed by measurement of transepithelial electrical resistance (TER). RESULTS: NPU cell cultures grew as monolayers in low-calcium, serum-free medium. Supplementation with 5% FBS and/or physiological calcium resulted in stratification into basal, intermediate, and superficial cell zones. Superficial cells were positive for UPIIIa, CK20, and ZO-1. TER measurement showed that NPU cells grown with FBS had significantly enhanced barrier function (6,720 ohms.cm(2)+/-1312 SD) compared with cells grown without FBS (102 ohms.cm(2)+/-34 SD; p<0.001). LIMITATIONS: Importantly, our study demonstrates that expression of differentiation-associated immunohistochemical markers by cultured urothelial cells can be regarded as evidence of only morphological differentiation and does not represent a surrogate marker of function. CONCLUSIONS: We have shown that normal porcine bladder urothelium has many cell biological properties equivalent to normal human urothelium, making it an excellent research substitute for difficult-to-obtain tissue. A differentiated, functional barrier urothelium has been produced from porcine bladder urothelial cells propagated in vitro and displays molecular and functional properties equivalent to native urothelium. This tissue has application in developing tissue-engineered bladders with urinary barrier properties and as a research tool for understanding the relationship between molecular and functional tissue differentiation.

Mutation analyses of Uroplakin II in children with renal tract malformations.

BACKGROUND: Uroplakin (UP) proteins cover urothelial apical surfaces. Mice lacking UPIIIa have elevated urothelial permeability and congenital renal tract anomalies, and UPIIIa mutations have been reported in children with kidney and ureter malformations. Mice with null mutation of another UP family member, UPII, are often born with congenital hydronephrosis. We hypothesized that UPII mutations may be present in humans with renal tract malformations. METHODS: Mutations were sought, using direct sequencing of the five UPII exons, in 42 children with diverse renal tract anomalies. RESULTS: No UPII abnormalities were detected in 41 patients, whereas one index case had a heterozygous frameshift change which, if expressed, would generate a truncated protein. This Caucasian child presented with vesicoureteric reflux (VUR), bilateral nephropathy and renal failure. The genetic change was also found in the index case's mother who had normal renal ultrasonography, but it was absent in 150 healthy Caucasian control individuals (96 assessed by direct sequencing and another 54 assessed by restriction digests). UPII was immunolocalized in urothelium of the normal human embryonic renal pelvis in a pattern similar to UPIIIa. CONCLUSION: This study offers no definitive support for UPII mutations causing human renal tract malformations. In rare patients, UPII variants might be implicated in pathogenesis when acting in conjunction with other yet-to-be-defined, genetic or environmental modifying factors.

Urothelial differentiation in vesicoureteric reflux and other urological disorders of childhood: a comparative study.

OBJECTIVE: The strong familial basis of vesicoureteric reflux (VUR) is well recognised, however little progress has been made in identifying the causative genes. In this study we have investigated whether human vesicoureteric reflux (VUR) is associated with the aberrant expression of urothelial differentiation-associated antigens in view of the VUR phenotype of the Uroplakin IIIa (UPIIIa) "knockout" mouse. METHODS: Samples of urothelium were collected from 13 children with primary VUR, four children with secondary VUR and from seven children with non-refluxing disorders of the urinary tract. Immunohistochemistry was used to assess the expression of five uroplakin (UP) and cytokeratin (CK) differentiation-restricted antigens, UPIa, UPIb, UPIIIa, CK13 and CK20. Samples were ranked blind according to immunohistochemical patterns relating to the differentiation-associated distributions of the five antigens and the results were analysed statistically using the Kruskal-Wallis test. RESULTS: No objective differences in urothelial morphology or the expression of the five differentiation antigens were discernable in the urothelium of children with primary VUR, when compared with urothelium of children with a range of other pathology including VUR associated with duplication or pelvic renal ectopia, VUR secondary to outflow obstruction and non-refluxing upper tract obstruction. The p-values ranged from 0.168-0.651 and were not considered statistically significant. CONCLUSION: The results indicate that primary VUR is not associated with any major, collective abnormality of urothelial differentiation in man. In particular our findings provide no support for the suggestion that abnormalities of UPIIIa expression are implicated in the aetiology of human primary VUR.

De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure.

Human renal adysplasia usually occurs sporadically, and bilateral disease is the most common cause of childhood end-stage renal failure, a condition that is lethal without intervention using dialysis or transplantation. De novo heterozygous mutations in Uroplakin IIIa (UPIIIa) are reported in four of 17 children with kidney failure caused by renal adysplasia in the absence of an overt urinary tract obstruction. One girl and one boy in unrelated kindreds had a missense mutation at a CpG dinucleotide in the cytoplasmic domain of UPIIIa (Pro273Leu), both of whom had severe vesicoureteric reflux, and the girl had persistent cloaca; two other patients had de novo mutations in the 3' UTR (963 T-->G; 1003 T-->C), and they had renal adysplasia in the absence of any other anomaly. The mutations were absent in all sets of parents and in siblings, none of whom had radiologic evidence of renal adysplasia, and mutations were absent in two panels of 192 ethnically matched control chromosomes. UPIIIa was expressed in nascent urothelia in ureter and renal pelvis of human embryos, and it is suggested that perturbed urothelial differentiation may generate human kidney malformations, perhaps by altering differentiation of adjacent smooth muscle cells such that the metanephros is exposed to a functional obstruction of urine flow. With advances in renal replacement therapy, children with renal failure, who would otherwise have died, are surviving to adulthood. Therefore, although the mechanisms of action of the UPIIIa mutations have yet to be determined, these findings have important implications regarding genetic counseling of affected individuals who reach reproductive age.