A study of cytokeratin 20 immunostaining in the urothelium of neuropathic bladder of patients with spinal cord injury

BACKGROUND: Normal urothelium is characterised by terminally differentiated superficial cells, which express cytokeratin 20 in the cytoplasm. In contrast, cultured human stratified urothelium, which does not undergo complete terminal differentiation of its superficial cells, does not express cytokeratin 20. If spinal cord injury (SCI) affects urothelial differentiation or induces squamous or other metaplastic change undetected by histological analysis, the superficial urothelial cells of the neuropathic bladder might be expected to show absence of immunostaining for cytokeratin 20. PATIENTS AND METHODS: We studied immunostaining for cytokeratin 20 in bladder biopsies taken from 63 consecutive SCI patients. Immunostaining was performed on paraffin-embedded tissue using a mouse monoclonal antibody (clone: Ks20.8). RESULTS: Of 63 biopsies, the epithelium was scarce in two. Eight biopsies showed squamous metaplasia and immunostaining for cytokeratin 20 was absent in all the eight biopsies. Of the remaining 53 cases, in which the umbrella cell layer of the urothelium was intact, immunostaining for cytokeratin 20 was seen only in ten biopsies. CONCLUSION: Superficial cells in the transitional epithelium showed immunostaining for cytokeratin 20 in 10 of 53 bladder biopsies taken from SCI patients. The reasons for this could be either that there is an underlying metaplasia or that changes in the neuropathic bladder affect urothelial differentiation. Taken with evidence from other systems, such as loss of cytokeratin 20 expression from static organ cultures of urothelial tissue, this might suggest that other factors, such as impairment of voluntary voiding in SCI patients, could affect expression of markers such as cytokeratin 20

Immunocytochemical characterisation of cultures of human bladder mucosal cells

<p>Abstract</p> <p>Background</p> <p>The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation.</p> <p>Methods</p> <p>Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence.</p> <p>Results</p> <p>Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections.</p> <p>Conclusions</p> <p>Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.</p

An ex Vivo

Transurothelial drug delivery continues to be an attractive treatment option for a range of urological conditions; however, dosing regimens remain largely empirical. Recently, intravesical delivery of the nonsteroidal anti-inflammatory ketorolac has been shown to significantly reduce ureteral stent-related pain. While this latest development provides an opportunity for advancing the management of stent-related pain, clinical translation will undoubtedly require an understanding of the rate and extent of delivery of ketorolac into the bladder wall. Using an ex vivo porcine model, we evaluate the urothelial permeability and bladder wall distribution of ketorolac. The subsequent application of a pharmacokinetic (PK) model enables prediction of concentrations achieved in vivo. Ketorolac was applied to the urothelium and a transurothelial permeability coefficient (Kp) calculated. Relative drug distribution into the bladder wall after 90 min was determined. Ketorolac was able to permeate the urothelium (Kp = 2.63 × 10–6 cm s–1), and after 90 min average concentrations of 400, 141 and 21 μg g–1 were achieved in the urothelium, lamina propria and detrusor respectively. An average concentration of 87 μg g–1 was achieved across the whole bladder wall. PK simulations (STELLA) were then carried out, using ex vivo values for Kp and muscle/saline partition coefficient (providing an estimation of vascular clearance), to predict 90 min in vivo ketorolac tissue concentrations. When dilution of the drug solution with urine and vascular clearance were taken into account, a reduced ketorolac concentration of 37 μg g–1 across the whole bladder wall was predicted. These studies reveal crucial information about the urothelium’s permeability to agents such as ketorolac and the concentrations achievable in the bladder wall. It would appear that levels of ketorolac delivered to the bladder wall intravesically would be sufficient to provide an anti-inflammatory effect. The combination of such ex vivo data and PK modeling provides an insight into the likelihood of achieving clinically relevant concentrations of drug following intravesical administration