Kees Jansen

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.

Biomarkers for the diagnosis of prostatic inflammation in benign prostatic hyperplasia

Chronic prostatic inflammation could be a central mechanism in benign prostatic hyperplasia (BPH) progression. Currently, the histological examination of prostate biopsies remains the only way to diagnose prostatic inflammation. Our objective was to find new noninvasive biomarkers for the diagnosis of prostatic inflammation.

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