Dick Janssen

The Mechanoreceptor TRPV4 is Localized in Adherence Junctions of the Human Bladder Urothelium: A Morphological Study

PURPOSE TRPV4 (transient receptor potential vanilloid 4 channel) is a nonselective cation channel involved in different sensory functions that was recently implicated in bladder mechanosensation. We investigated the cellular site of TRPV4 in bladder urothelium and explored a molecular connection between TRPV4 and urothelial adherence junctions. MATERIALS AND METHODS We obtained healthy tissues sections from cystectomy in humans due to cancer in 3 and noncancerous conditions in 2. Besides human biopsies tissues from 7 normal and 7 TRPV4-/-mice, and the urothelial cell line RT4 were also used. Experiments were done with polyclonal antibody against TRPV4 (against the N-terminus of rat TRPV4). A molecular connection between TRPV4 and different adherence junction components was investigated using immunofluorescence, Western blot and immunoprecipitation. RESULTS Results revealed TRPV4 on urothelial cell membranes near adherence junctions. Results were comparable in the urothelial cell line, human bladders and mouse bladders. Subsequent immunoprecipitation experiments established a molecular connection of TRPV4 to α-catenin, an integral part of the adherence junction that catenates E-cadherin to the actin-microfilament network. CONCLUSIONS Results provide evidence for the location of TRPV4 in human bladder urothelium. TRPV4 is molecularly connected to adherence junctions on the urothelial cell membrane. TRPV4 coupling to a rigid intracellular and intercellular structural network would agree with the hypothesis that TRPV4 can be activated by bladder stretch.

The Distribution and Function of Chondroitin Sulfate and Other Sulfated Glycosaminoglycans in the Human Bladder and Their Contribution to the Protective Bladder Barrier

PURPOSE Glycosaminoglycan replenishment therapies are commonly applied to treat bladder inflammatory conditions such as bladder pain syndrome/interstitial cystitis. Although there is evidence that these therapies are clinically effective, much is still unknown about the location and function of different types of glycosaminoglycans in the bladder. We investigated the location of sulfated glycosaminoglycans in the bladder and evaluated their contribution to the urothelial barrier. MATERIALS AND METHODS The location of different glycosaminoglycans (heparan sulfate, chondroitin sulfate and dermatan sulfate) in human and porcine bladders was investigated with immunofluorescence staining and isolating glycosaminoglycans using selective urothelial sampling techniques. Barrier function was evaluated with transepithelial electrical resistance measurements (Ω.cm(2)) on primary porcine urothelial cell cultures. The contribution of different glycosaminoglycans to the bladder barrier was investigated with specific glycosaminoglycan digesting enzymes and protamine. RESULTS High glycosaminoglycan concentrations are located around the urothelial basal membrane and at the urothelial luminal surface. After removing the glycosaminoglycan layer, urothelial permeability increased. Natural recovery of the glycosaminoglycan layer takes less than 24 hours. Chondroitin sulfate was the only sulfated glycosaminoglycan that was located on the urothelial luminal surface and that contributed to urothelial barrier function. CONCLUSIONS This study reveals an important role for chondroitin sulfate in bladder barrier function. Therapies aiming at restoring the luminal glycosaminoglycan layer in pathological conditions such as bladder pain syndrome/interstitial cystitis are based on a sound principle.

Urgent-SQ implant in treatment of overactive bladder syndrome: 9-year follow-up study†‡

Electrical stimulation of the posterior tibial nerve (PTN) is an established therapy for the treatment of refractory overactive bladder syndrome (OAB). The Urgent‐SQ™ is an implant that is surgically placed near the PTN and activated by an external pulse generator, allowing for “on demand” PTN stimulation, without the need for needle insertion. The current study presents results of a 9‐year, open‐label, follow‐up of eight patients to address the long term safety and efficacy of the implant.

Urothelium update: how the bladder mucosa measures bladder filling

This review critically evaluates the evidence on mechanoreceptors and pathways in the bladder urothelium that are involved in normal bladder filling signalling.

TRPV4 channels in the human urogenital tract play a role in cell junction formation and epithelial barrier

The molecular interactions between transient receptor potential vanilloid subtype 4 channels (TRPV4) and cell junction formation were investigated in the human and mouse urogenital tract.

A New, Straightforward Ex Vivo Organoid Bladder Mucosal Model for Preclinical Research

PURPOSE We developed an experimental ex vivo organoid bladder mucosal model that can be used for experimental research purposes to create alternatives to current animal models. MATERIALS AND METHODS We developed an ex vivo organoid bladder mucosal model by immobilizing a type I collagen scaffold on the bottom of a Transwell® insert, creating a 2-compartment system. Mucosal biopsies from porcine bladders were placed on top of the scaffold and cultured in different mediums. We evaluated the morphological aspects of biopsy tissue. Cultured samples were assessed by scanning electron microscopy, and immunohistochemical and histochemical staining for cell identification, proliferation and morphology. RESULTS Cells remained viable in Dulbeccos modified Eagles medium/F-12 and smooth muscle cell medium for up to 3 weeks. The mucosa retained normal morphological characteristics for up to 1 week. Cells (mostly urothelial cells) proliferated and fully covered the scaffold surface within 3 weeks. CONCLUSIONS We developed an experimental ex vivo organoid model of bladder mucosa for preclinical experimental research. This model could be used for high volume screening for pharmacology and toxicology experiments. It has the potential to replace currently used animal models.

Clinical utility of neurostimulation devices in the treatment of overactive bladder: current perspectives

Objectives This review describes the evidence from established and experimental therapies that use electrical nerve stimulation to treat lower urinary tract dysfunction. Methods Clinical studies on established treatments such as percutaneous posterior tibial nerve stimulation (P-PTNS), transcutaneous electrical nerve stimulation (TENS), sacral nerve stimulation (SNS) and sacral anterior root stimulation (SARS) are evaluated. In addition, clinical evidence from experimental therapies such as dorsal genital nerve (DGN) stimulation, pudendal nerve stimulation, magnetic nerve stimulation and ankle implants for tibial nerve stimulation are evaluated. Results SNS and P-PTNS have been investigated with high-quality studies that have shown proven efficacy for the treatment for overactive bladder (OAB). SARS has proven evidence-based efficacy in spinal cord patients and increases the quality of life. TENS seems inferior to other OAB treatments such as SNS and P-PTNS but is noninvasive and applicable for ambulant therapy. Results from studies on experimental therapies such as pudendal nerve stimulation seem promising but need larger study cohorts to prove efficacy. Conclusion Neurostimulation therapies have proven efficacy for bladder dysfunction in patients who are refractory to other therapies. Significance Refinement of neurostimulation therapies is possible. The aim should be to make the treatments less invasive, more durable and more effective for the treatment of lower urinary tract dysfunction.

257 TRPV4 IS INVOLVED IN CELL JUNCTION FORMATION IN THE UROGENITAL TRACT. AN ULTRASTRUCTURAL STUDY

Fig 2) Immunofluorescence stainings on wildtype (WT; A,B,C,D,I) and TRPV4 -/(E,F,G,H,J) mice bladder (A-H) and kidney (I,J) tissue using antibodies against TRPV4 (A,F) and AJ’s (Ecadherin; E-cad (B,D,E,G,I ,J). Images C & H show merged image. • TRPV4 co localizes with AJ’s in WT mouse (A-C). No TRPV4 signal was detected in TRPV4 -/mouse tissue (F). • WT mouse bladders show distinct formation of AJ’s between connecting urothelial cells (B,D; arrow). • In TRPV4 -/mice, AJ formation is reduced and intercellular spaces are larger (G,E). • This pattern also is seen in the distal collecting ducts of the kidney, where there are less cell junction ridges (AJ’s) visible in the TRPV4 -/mice (J) compared to WT mice (I) (arrows). Fig 4) A, B, C: TEM imaging of wild type bladder with normal urothelium. A) overview, B) normal tight junction (red arrow), C) normal AJ’s (orange arrow) & desmosomes (blue arrow), D, E, F : TEM imaging of TRPV4 -/mouse bladder urothelium. E) overview with enlarged intercellular spaces between cells (green arrow), F) tight junction (red arrow), G) desmosomes (blue arrow), but no AJ’s present. •TRPV4 co localizes with adherence junctions throughout the urogenital