Melatonin was recently shown to have protective effects against cyclophosphamide (CP)-induced haemorrhagic cystitis (HC) by diminishing bladder oxidative stress. HC is accompanied by destruction of the bladder urothelium and followed by apoptosis and rapid regeneration via proliferation and differentiation of urothelial cells, reaching complete restoration of normal urothelium in three weeks. Therefore, the effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells, during destruction and regeneration of the urothelium three-weeks after a single dose of CP treatment, was studied. F344 male rats were injected intraperitoneally with saline (control group) or melatonin (Mel group) or a single dose of CP (100 mg/kg; CP group) or melatonin (10 mg/kg) with CP (Mel + CP group). Melatonin co-treatment with CP significantly reduced apoptosis and increased proliferation of urothelial cells at day 1 and thus prevented extensive loss of cells from the urothelium. However, proliferation indices at days 4 and 7 after melatonin and CP co-treatment suddenly dropped and therefore the development of hyperplasia was prevented. Melatonin co-treatment with CP also resulted in earlier differentiation of superficial urothelial cells. Melatonin seems to have protective effect against CP-induced urothelial damage and a favourable impact on regeneration and restoration of normal urothelium, since it reduces the number of apoptotic and proliferating urothelial cells and results in their earlier differentiation.
COBISS.SI-ID: 24044505
The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5-15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.
COBISS.SI-ID: 25479641
Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal or fusiform-shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three-dimensional localization using confocal microscopy of immunofluorescence-labelled GA-related markers šGM130 (cis-Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin, uroepithelial differentiation-related markers (UPs), MTs (microtubules; á-tubulin) and intermediate filaments šCK7 (cytokeratin 7) and CK20đ, we found that in non-differentiated, UP-negative UCs the GA is mostly organized as a single ribbon-like structure close to the nucleus, whereas in differentiated, UP-positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans-Golgi/TGN enzyme â1,4-galactosyltransferase) fused to fluorescent protein showed that Golgi-resident enzyme cycles freely within ribbon-like GA but not within fragmented GA. By CLEM (correlative light-electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. (Abstract truncated at 2000 characters)
COBISS.SI-ID: 27453657
In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs co-localize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.
COBISS.SI-ID: 29577177
The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 microm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.
COBISS.SI-ID: 30076889
When the urothelial barrier, i.e., the blood-urine barrier, is injured, rapid resealing of the injury is crucial for the normal functioning of the organism. In order to investigate the mechanisms required for rapid resealing of the barrier, we established in vitro models of hyperplastic and normoplastic urothelia. We found that hyperplastic urothelia achieve significantly higher transepithelial resistance (TER) than normoplastic urothelia. However, the expression of cell junctional (claudin-8, occludin, E-cadherin) and differentiation-related proteins (cytokeratin 20 and uroplakins) is weaker in hyperplastic urothelia. Further investigation of cell differentiation status at the ultrastructural level confirmed that superficial urothelial cells (UCs) in hyperplastic urothelial models achieve a lower differentiation stage than superficial UCs in normoplastic urothelial models. With the establishment of such in vitro models and the aid of TER measurements, flow cytometry, molecular and ultrastructural analysis, we here provide unequivocal evidence that the specific cell-cycle distribution and, consequently, the number of cell layers have a significant influence on the barrier function of urothelia. We demonstrate the importance of hyperplasia for the rapid restoration of the urothelial barrier and the maintenance of high TER until the UCs reach a highly differentiated stage and restoration of the urothelial barrier after injury is complete. The information that this approach provides is unique and we expect that further exploitation of hyperplastic and normoplastic urothelial models in future studies may advance our understanding of blood-urine barrier development and functionality.
COBISS.SI-ID: 29338585
Background: Urothelial bladder is the reservoir of urine and the urothelium minimizes the exchange of urine constituents with this tissue. Our aim was to test 1.9 nm biocompatible gold nanoparticles as a novel marker of internalization into the urothelial cells under physiological conditions in vivo. Methods: We compared normal and neoplastic mice urothelium. Neoplastic lesions were induced by 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water for 10 weeks. Nanoparticles, intravenously injected into normal and BBN-treated mice, were filtered through the kidneys and became constituents of the urine within 90 minutes after injection. Results: Gold nanoparticles were densely accumulated in the urine, while their internalization into urothelial cells depended on the cell differentiation stage. In the terminally differentiated superficial urothelial cells of normal animals, nanoparticles were occasionally found in the endosomes, but not in the fusiform vesicles. Regions of exfoliated cells were occasionally found in the normal urothelium. Superficial urothelial cells located next to exfoliated regions contained gold nanoparticles in the endosomes and in the cytosol beneath the apical plasma membrane. The urothelium of BBN-treated animals developed flat hyperplasia with moderate dysplasia. The superficial cells of BBN-treated animals were partially differentiated as demonstrated by the lack of fusiform vesicles. These cells contained the gold nanoparticles distributed in the endosomes and throughout their cytosol. Conclusion: Gold nanoparticles are a valuable marker to study urine internalization into urothelial cells in vivo. Moreover, they can be used as a sensitive marker of differentiation and functionality of urothelial cells.
COBISS.SI-ID: 30967257
High transepithelial electrical resistance (TEER) demonstrates a functional permeability barrier of the normal urothelium, which is maintained by a layer of highly differentiated superficial cells. When the barrier is challenged, a quick regeneration is induced. We used side-by-side diffusion chambers as an ex vivo system to determine the time course of functional and structural urothelial regeneration after chitosan-induced injury. The exposure of the urothelium to chitosan caused a 60 % decrease in TEER, the exposure of undifferentiated urothelial cells to the luminal surface and leaky tight junctions. During the regeneration period (350 min), TEER recovered to control values after approximately 200 min, while structural regeneration continued until 350 min after injury. The tight junctions are the earliest and predominant component of the barrier to appear, while complete barrier regeneration is achieved by delayed superficial cell terminal differentiation. The barrier function and the structure of untreated urothelium were unaffected in side-by-side diffusion chambers for at least 6 h. The urinary bladder tissue excised from an animal thus retains the ability to maintain and restore the transepithelial barrier and cellular ultrastructure for a sufficient period to allow for studies of regeneration in ex vivo conditions.
COBISS.SI-ID: 30654169
The primary function of the urinary bladder is to store and periodically release urine. How the urothelium prevents permeation of water, ions, solutes, and noxious agents back into the bloodstream and underlying tissues as well as serving as a sensor and transducer of physiological and nociceptive stimuli is still not completely understood, and thus its unique functional complexity remains to be fully elucidated. This article reviews the permeation routes across urothelium as demonstrated in extensive morphological and electrophysiological studies on in vivo and in vitro urothelia. We consider the molecular and morphological structures of urothelium and how they contribute to the impermeability of the blood-urine barrier. Based on the available data, the extremely low permeability properties of urothelium can be postulated. This remarkable impermeability is necessary for the normal functioning of all mammals, but at the same time represents limitations regarding the uptake of drugs. Therefore, the current progress to overcome this most resilient barrier in our body for drug therapy purposes is also summarized in this review.
COBISS.SI-ID: 31938777
Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, colonize and invade the epithelial cells of the bladder urothelium. Infection of immature urothelial cells can result in the formation of persistent intracellular reservoirs that are refractory to antibiotic treatments. Previously, we defined a novel therapeutic strategy utilizing the bladder cell exfoliant chitosan to deplete UPEC reservoirs. However, though a single treatment of chitosan followed by ciprofloxacin administration had a marked effect on reducing UPEC titers within the bladder, this treatment failed to prevent relapsing bacteriuria. We show here that repeated application of chitosan in conjunction with the antibiotic ciprofloxacin, completely eradicates UPEC from the urinary tract and prevents the development of relapsing bouts of bacteriuria. In addition, microscopy revealed rapid restoration of bladder integrity following chitosan treatment, indicating that chitosan can be used to effectively combat recalcitrant bladder infections without causing any lasting harm to the urothelium. The potential therapeutic value of combinatorial treatments with chitosan and antibiotics like ciprofloxacin requires further investigation, but the findings presented here provide a strong impetus for pursuing this approach as an option for individuals who have chronic UTIs that are unresolved by use of more classical antibiotic therapies.
COBISS.SI-ID: 33123801