Jennifer Hinley

PPARγ-Regulated Tight Junction Development During Human Urothelial Cytodifferentiation

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation‐associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1–5 and 7. Following induction of differentiation by pharmacological activation of PPARγ and blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation‐associated development of TJ formation in vitro reflected the stage‐related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARγ and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme. J. Cell. Physiol. 208: 407–417, 2006.

Differentiation Potential of Urothelium from Patients with Benign Bladder Dysfunction

To develop a novel in vitro approach to test the hypothesis that failure of urothelial differentiation underlies the aetiopathology of interstitial cystitis (IC), where there is evidence of compromised urinary barrier function, as benign dysfunctional bladder disease encompass several poorly understood clinically defined conditions, including IC, idiopathic detrusor overactivity (IDO) and stress urinary incontinence (SUI).

Tissue engineering potential of urothelial cells from diseased bladders.

PURPOSE We examined the suitability of urothelium from patients with abnormal bladders for use in surgical reconstruction using a tissue engineering approach that would require autologous urothelium to be expanded by propagation in cell culture. MATERIALS AND METHODS Resection specimens from 8 children (median age 9.8 years) with abnormal bladders (neuropathic in 4, posterior urethral valves in 2, epispadias in 1, nonneurogenic in 1) were collected with informed parental consent during planned urological procedures. Six patients had recurrent urinary tract infections and 7 underwent frequent intermittent catheterization. A representative sample was immunohistologically processed to assess urothelial proliferation and differentiation status, and the remaining 7 cases were processed for urothelial cell culture. Five normal adult urothelial samples were included as controls. RESULTS Immunohistological assessment indicated that 3 of 8 samples lacked urothelial differentiation associated expression of UPK3a or CK20. Four of 7 samples resulted in successful primary culture, with 1 sample lost to underlying infection and 2 not surviving in culture. All 4 cultures grew beyond passage 3 before senescence but all showed reduced proliferation capacity and a compromised ability to form a barrier urothelium compared to controls. CONCLUSIONS While normal human urothelium is highly regenerative and derived cells are highly proliferative in culture, our results with urothelium from abnormal pediatric bladders indicate a reduced capacity for proliferation and differentiation in vitro. This finding may indicate a need to identify alternative cell sources for engineered bladder reconstruction.

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.

Identification of ELF3 as an early transcriptional regulator of human urothelium

Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes. However, unless datasets are exactly synchronous, time points cannot be compared directly. Here we present a data-driven analysis of regulatory elements from a microarray time series that tracked the differentiation of non-immortalised normal human urothelial (NHU) cells grown in culture. The datasets were obtained by harvesting differentiating and control cultures from finite bladder- and ureter-derived NHU cell lines at different time points using two previously validated, independent differentiation-inducing protocols. Due to the asynchronous nature of the data, a novel ranking analysis approach was adopted whereby we compared changes in the amplitude of experiment and control time series to identify common regulatory elements. Our approach offers a simple, fast and effective ranking method for genes that can be applied to other time series. The analysis identified ELF3 as a candidate transcriptional regulator involved in human urothelial cytodifferentiation. Differentiation-associated expression of ELF3 was confirmed in cell culture experiments and by immunohistochemical demonstration in situ. The importance of ELF3 in urothelial differentiation was verified by knockdown in NHU cells, which led to reduced expression of FOXA1 and GRHL3 transcription factors in response to PPARγ activation. The consequences of this were seen in the repressed expression of late/terminal differentiation-associated uroplakin 3a gene expression and in the compromised development and regeneration of urothelial barrier function.

Nerve hyperplasia: a unique feature of ketamine cystitis

BackgroundThere is an emerging association between ketamine abuse and the development of urological symptoms including dysuria, frequency and urgency, which have a neurological component. In addition, extreme cases are associated with severe unresolving bladder pain in conjunction with a thickened, contracted bladder and an ulcerated/absent urothelium. Here we report on unusual neuropathological features seen by immunohistology in ketamine cystitis.ResultsIn all cases, the lamina propria was replete with fine neurofilament protein (NFP+) nerve fibres and in most patients (20/21), there was prominent peripheral nerve fascicle hyperplasia that showed particular resemblance to Morton’s neuroma. The nerve fascicles, which were positive for NFP, S100 and the p75 low-affinity nerve growth factor receptor (NGFR), were generally associated with a well-developed and in places, prominent, epithelial membrane antigen+/NGFR+ perineurium. This peripheral nerve fascicle hyperplasia is likely to account for the extreme pain experienced by ketamine cystitis patients. Urothelial damage was a notable feature of all ketamine cystitis specimens and where urothelium remained, increased NGFR expression was observed, with expansion from a basal-restricted normal pattern of expression into the suprabasal urothelium.ConclusionsThe histological findings were distinguishing features of ketamine cystitis and were not present in other painful bladder conditions. Ketamine cystitis afflicts predominantly young patients, with unknown long-term consequences, and requires a strategy to control severe bladder pain in order to remove a dependency on the causative agent. Our study indicates that the development of pain in ketamine cystitis is mediated through a specific neurogenic mechanism that may also implicate the urothelium.

Trans-Species Comparison of PPAR and RXR Expression by Rat and Human Urothelial Tissues

Because some investigational peroxisome proliferator-activated receptors (PPAR) agonists cause tumors in the lower urinary tract of rats, we compared normal human and rat urothelium in terms of PPAR and retinoid X receptor (RXR) expression and proliferation-associated phenotypes. In situ, few human but most rat urothelial cells were Ki67 positive, indicating fundamental differences in cell cycle control. Rat and human urothelia expressed all 3 PPAR and the RXRα and RXRβ isoforms in a predominantly nuclear localization, indicating that they may be biologically active. However, immunolocalization differences were observed between species. First, whereas PPARα and PPARβ/δ were expressed throughout the human bladder or ureteric urothelium, in the rat urothelium PPARα was primarily, and PPARβ/δ exclusively, restricted to superficial cells. Second, RXRβ was restricted to intermediate and superficial layers of the human urothelium but tended to be absent from the rat superficial cells. Third, PPARγ expression was present throughout the urothelia of both species but was most intense in the superficial human urothelium. Species differences were also observed in the expression of PPAR and RXR isoforms between cultured rat and human urothelial cells and in the smooth muscle. Our findings highlight the unique coexpression of multiple PPAR and RXR isoforms by urothelium and suggest that species differences in PPAR function between rat and human urothelia may be explored in an in vitro setting.

Use of donor bladder tissues for in vitro research.

To evaluate deceased non‐heart beating (DNHB) donors and deceased heart beating (DHB) brain‐stem dead donors, as sources of viable urological tissue for use in biomedical research. To identify sources of viable human bladder tissue as an essential resource for cell biological research aimed at understanding human diseases of the bladder and for developing new tissue engineering and regenerative medicine strategies for bladder reconstruction. Typically, normal human urinary tract tissue is obtained from adult or paediatric surgical patients with benign urological conditions, but few surgical procedures yield useful quantities of healthy bladder tissue for research.

The human urothelial tight junction: claudin 3 and the ZO-1α(+) switch.

Objective Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional “tightness” of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ. Materials and Methods Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+ was examined in native urothelium by immunohistochemistry. Results Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+ was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+ isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α−/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function. Conclusions Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+ isotype was also observed and for the first time may indicate that ZO-1α+ is involved in the structural assembly and function of the urothelial terminal TJ.

PPARα and PPARγ Coactivation Rapidly Induces Egr-1 in the Nuclei of the Dorsal and Ventral Urinary Bladder and Kidney Pelvis Urothelium of Rats

To facilitate studies of the rat bladder carcinogenicity of dual-acting PPARα+γ agonists, we previously identified the Egr-1 transcription factor as a candidate carcinogenicity biomarker and developed rat models based on coadministration of commercially available specific PPARα and PPARγ agonists. Immunohistochemistry for Egr-1 with a rabbit monoclonal antibody demonstrated that male vehicle-treated rats exhibited minimal urothe-lial expression and specifically, no nuclear signal. In contrast, Egr-1 was induced in the nuclei of bladder, as well as kidney pelvis, urothelia within one day (2 doses) of oral dosing of rats with a combination of 8 mg/kg rosiglitazone and 200 mg/kg fenofibrate (specific PPARγ and PPARα agonists, respectively). These findings were confirmed by Western blotting using a different Egr-1 antibody. Egr-1 was induced to similar levels in the dorsal and ventral bladder urothelium, arguing against involvement of urinary solids. Egr-1 induction sometimes occurred in a localized fashion, indicating physiological microheterogeneity in the urothelium. The rapid kinetics supported that Egr-1 induction occurred as a result of pharmacological activation of PPARα and PPARγ, which are coexpressed at high levels in the rat urothelium. Finally, our demonstration of a nuclear localization supports that the Egr-1 induced by PPARα and PPARγ coactivation in the rat urothelium may be biologically active.