Aquaporin 3 Expression Loss in Urothelial Carcinoma: Association with Tumor Invasion Depth, but not with Grading?

We have previously provided molecular evidence of expression of aquaporin 3 (AQP3) in normal human urothelium and in UBC of various stages. Whereas former studies demonstrated that loss of AQP3 was associated with invasive and high-grade disease and worse progression-free and cancer-specific survival, this report investigates the expression of AQP3 in associated CIS. Contrary to what we had expected, all CIS specimens were shown to exhibit strong AQP3 expression, suggesting loss of AQP3 in UBC is primarily associated with the ability of tumor cells for invasion but not with grading as sign of dedifferentiation.

Continent Anal Urinary Diversion in Classic Bladder Exstrophy: 45-Year Experience.

OBJECTIVE: To evaluate the long-term outcomes in patients with classic bladder exstrophy and continent anal urinary diversion (CAD) for continence, upper urinary tract status, secondary malignancies, and sexual function. PATIENTS AND METHODS: The medical records of 82 exstrophy patients having undergone CAD in our department between 1970 and 2015 were reviewed. Patients were invited for follow-up examinations and asked to complete validated questionnaires relating to sexual function. RESULTS: Thirty-two of 57 eligible patients with a median follow-up of 23.9 years were included in the study. Ninety-seven percent of patients were fully continent during daytime. Upper urinary tract and renal function remained stable in 75% and 87%, respectively. Five patients developed secondary malignancies originating from the rectal reservoir. Forty-one percent received prophylactic alkaline substitution. Sexual function as measured by the Female Sexual Function Index and the International Index on Erectile Function was negatively affected in all domains in both genders. Eighty-six percent of patients had a stable relationship and 35% were married. Five women conceived a total of 6 healthy children. Paternity rate was 40%. CONCLUSION: CAD constitutes an effective treatment option with acceptable long-term outcomes in exstrophy patients in whom all attempts at restoring the lower urinary tract have failed. Long-term follow-up of the upper urinary tract, assessment of acid-base balance, and endoscopy of the rectosigmoid reservoir are paramount for the safety of this type of management. Evaluation of sexual dysfunction should be an active part of follow-up.

Expression of aquaporin water channels in human urothelial carcinoma: correlation of AQP3 expression with tumour grade and stage.

PURPOSE: To study the expression, localization and potential clinical significance of aquaporin water channels both in well-established urothelial cancer (UC) cell lines and in human bladder carcinoma specimens of different stages and grades and to discuss the clinical relevance of the findings. METHODS: AQP transcript and protein expression by RT4, RT112 and T24 UC cell lines was investigated using reverse transcriptase polymerase chain reaction and immunofluorescence labelling. Immunohistochemistry (IHC) was used to assess AQP protein expression in 94 UC specimens of various grades and stages. RESULTS: AQP3 and 9 transcripts were expressed in low-grade RT4 and RT112, but not in high-grade T24 cells. By contrast, AQP4 mRNA was absent in RT4, but expressed by RT112 and T24. Transcripts for AQP7 and 11 were detected in all three UC cell lines. Immunofluorescence microscopy confirmed the expression of AQP3, 4 and 7 at the protein level. By IHC, AQP3 was shown to be intensely expressed by 86 %, 66 % and 33 % of specimens of stage pTa, pT1 and pT2 tumours, respectively (p < 0.001). Whereas 100 % of G1 tumours were positive, only 73 % and 55 % of G2 and G3 tumours were found to express AQP3 (p = 0.004). CONCLUSIONS: This is the first study to demonstrate that several AQPs are expressed in UC. Our results indicate that there is a correlation between AQP3 protein expression and tumour stage and grade, with AQP3 expression being reduced or lost in tumours of higher grade and stage. Taken together with the available evidence from other studies, we conclude that AQPs may play a role in the progression of UC and, in particular, that this could be of prognostic value.

Delayed-onset ureteral obstruction after endoscopic dextranomer/hyaluronic acid copolymer (Deflux) injection for treatment of vesicoureteral reflux in children: a case series.

We report 4 patients with upper urinary tract (UUT) obstruction requiring ureteric reimplantation at 1, 7, 28, and 63 months after dextranomer/hyaluronic acid copolymer (Dx/HA) injection for vesicoureteric reflux. Histopathologic evaluation of ureteric segments revealed extensive foreign body formation in all cases. We conclude that UUT obstruction is a rare but serious complication after Dx/HA injection that can occur even years after surgery. The incidence of delayed-onset UUT obstruction may be higher than previously noted. Long-term follow-up and a critical reappraisal of the method are needed to assess the late sequelae of Dx/HA injection therapy for vesicoureteric reflux.

Persistent histological changes in the exstrophic bladder after primary closure-a cause for concern?

PURPOSE: We investigated bladder biopsies from patients with classic bladder exstrophy for the histological features and discuss the potential clinical significance of the findings. MATERIALS AND METHODS: Bladder tissues were collected from patients with bladder exstrophy between 2004 and 2011. These specimens were obtained at primary bladder closure (group 1, 29 patients), during secondary reconstructive procedures (group 2, 27) or during cystectomy for failed reconstruction (group 3, 15). All tissue specimens were investigated for inflammatory, proliferative, metaplastic and dysplastic changes. Expression of urothelial differentiation markers CK13 and CK20 was determined by immunohistochemical analysis. RESULTS: Inflammatory, proliferative and metaplastic changes were found in bladder specimens of all subgroups. Neither dysplasia nor neoplasia was present. Severe epithelial changes such as cystitis glandularis and intestinal metaplasia were observed in up to 62% of bladders several years after primary closure. Aberrant expression patterns of CK13 and CK20 suggesting abnormal urothelial differentiation were shown to be present in the urothelium of all subgroups. CONCLUSIONS: Our findings provide prima facie evidence that the epithelial changes observed in the unclosed bladder template persist or even progress in a subset of bladders after primary closure. Although the malignant potential of cystitis glandularis and intestinal metaplasia is controversial, some patients may be at increased risk for dysplasia/neoplasia in the long term. Since the natural history of these lesions in the exstrophic bladder is unknown, these patients require lifelong surveillance.

Aquaporin expression contributes to human transurothelial permeability in vitro and is modulated by NaCl.

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm(2)), with mean diffusive water and urea permeability coefficients of 6.33×10(-5) and 2.45×10(-5) cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.

Loss of aquaporin 3 protein expression constitutes an independent prognostic factor for progression-free survival: an immunohistochemical study on stage pT1 urothelial bladder cancer.

BACKGROUND: Treatment of patients with stage pT1 urothelial bladder cancer (UBC) continues to be a challenge due to its unpredictable clinical course. Reliable molecular markers that help to determine appropriate individual treatment are still lacking. Loss of aquaporin (AQP) 3 protein expression has previously been shown in muscle-invasive UBC. The aim of the present study was to investigate the prognostic value of AQP3 protein expression with regard to the prognosis of stage pT1 UBC. METHOD: AQP 3 protein expression was investigated by immunohistochemistry in specimens of 87 stage T1 UBC patients, who were diagnosed by transurethral resection of the bladder (TURB) and subsequent second resection at a high-volume urological centre between 2002 and 2009. Patients underwent adjuvant instillation therapy with Bacillus Calmette-Guérin (BCG). Loss of AQP3 protein expression was defined as complete absence of the protein within the whole tumour. Expression status was correlated retrospectively with clinicopathological and follow-up data (median: 31 months). Multivariate Cox regression analysis was used to assess the value of AQP3 tumour expression with regard to recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS). RFS, PFS and CSS were calculated by Kaplan-Meier analysis and Log rank test. RESULTS: 59% of patients were shown to exhibit AQP3-positive tumours, whereas 41% of tumours did not express the marker. Loss of AQP3 protein expression was associated with a statistically significantly worse PFS (20% vs. 72%, p=0.020). This finding was confirmed by multivariate Cox regression analysis (HR 7.58, CI 1.29 - 44.68; p=0.025). CONCLUSIONS: Loss of AQP3 protein expression in pT1 UBC appears to play a key role in disease progression and is associated with worse PFS. Considering its potential prognostic value, assessment of AQP3 protein expression could be used to help stratify the behavior of patients with pT1 UBC.

Expression and potential clinical significance of urothelial cytodifferentiation markers in the exstrophic bladder.

PURPOSE: We characterize the urothelium from patients with classic bladder exstrophy-epispadias complex for the expression of proteins associated with urothelial differentiation, and discuss a potential impact of urothelial phenotype on the structural and functional properties of the bladder template following bladder closure. MATERIALS AND METHODS: From 2005 to 2010 bladder biopsies from 32 infants with bladder exstrophy-epispadias complex obtained at primary bladder closure were collected. After histological assessment immunochemistry was used to investigate the expression of uroplakin IIIa, cytokeratin differentiation restricted antigens CK13 and CK20, and tight junction protein claudin 4. RESULTS: Overall tissue morphology showed gross alterations with inflammatory, proliferative and metaplastic changes in most specimens. Sections of intact epithelium were present in 78% of biopsies. With respect to urothelial phenotype, CK13 was expressed in all specimens, whereas UPIIIa and CK20 were absent in 76% of the tissues examined. Of the biopsies 52% revealed an irregular expression pattern of tight junction protein Cl-4. CONCLUSIONS: This is the first study to our knowledge to characterize the urothelium from infants with bladder exstrophy-epispadias complex for the expression of urothelial differentiation associated antigens. Our findings suggest urothelial differentiation changes in a majority of exstrophic bladders, at least at primary bladder closure. Although the underlying etiology remains to be established, abnormal urothelial differentiation may result in a dysfunctional urothelial barrier with implications for the structural and functional properties of the bladder template. Despite the study limitations, our preliminary findings provide a platform for further investigation of the significance of the urothelium for the exstrophic bladder.

15 years of continent urinary diversion and enterocystoplasty in children and adolescents: the Würzburg experience.

OBJECTIVE: To assess the long-term results of continent urinary diversion (CUD) and enterocystoplasty (ECP) in children with irreversible lower urinary tract dysfunction (LUTD). PATIENTS AND METHODS: The study included 44 children with irreversible LUTD who had a CUD or ECP between 1992 and 2007. Patients were followed for the functional outcome of surgery with a focus on complications related to the reservoir, bowel, uretero-intestinal anastomosis and upper urinary tract. Data were collected prospectively and outcomes were evaluated using a standardized protocol. RESULTS: The median (range) follow-up was 7.3 (3.5-17) years. Complete continence was achieved in 94% overall, i.e. in 95% of patients with continent cutaneous diversion, 83% with ECP and all children with continent anal diversion. Upper urinary tract and renal function remained stable in 89% and 95%, respectively. Surgical intervention was required for adhesive small bowel ileus in 6%, stoma-related complications in 39%, ureteric stenosis in 8%, and stone formation in 19%. Of these complications, 54% required only minor interventions; 41% of patients needed prophylactic alkaline substitution. Bowel habits remained unchanged or improved in 68%. CONCLUSION: Our results show that CUD and ECP in children are effective procedures with acceptable long-term complication rates. However, conclusions from our data might be limited, as this was a small study including highly selected patients treated at one tertiary academic centre. Being an audit of practice in our institution and given the variety of concepts, these results might differ from those centres using other approaches in the surgical treatment of LUTD. Importantly, this type of surgery should be restricted to carefully selected patients in whom all attempts of restoring the LUT failed.

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.