The complete functional recovery of chitosan-treated biomimetic hyperplastic and normoplastic urothelial models

Abstract

The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05%) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.

Co‐culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research

“…The immunofluorescence reaction was performed as described previously (Kreft et al, 2005b). Each antibody used in this study has been previously extensively tested in our lab by performing immunofluorescence as well as immunohistochemistry by biotinylated secondary antibodies on cell cultures and on cryo‐ and paraffin‐sections of pig, mouse, rat, and human urinary bladder urothelium tissue sections (unpublished research and Kreft et al, , , , , 2010; Visnjar et al, ; Visnjar and Kreft, , ; Zupancic et al, , , ; Zupancic and Romih, ). The panel of antibodies we used was: CK 7 (mouse monoclonal antibody, diluted 1:20, Dako, Glostrup, Denmark), vimentin (rabbit polyclonal antibody, diluted 1:20, Dako), desmin (rabbit polyclonal antibody, diluted 1:40, Sigma), collagen I (mouse monoclonal antibody, diluted 1:200, Sigma), occludin (rabbit polyclonal antibody, diluted 1:20, Zymed Laboratories, San Francisco, CA, USA), E‐cadherin (mouse monoclonal antibody, diluted 1:20, Transduction Laboratories, Lexington, KY, USA), uroplakins (rabbit polyclonal antibody, diluted 1:10.000, a kind gift from Prof. dr. T.T.…”

“…The second one is DMEM and Ham's F12 supplemented with 5%, 10%, or 20% fetal bovine serum (FBS) (Fujiyama et al, ; Ehmann and Terris, ; Fossum et al, ; Larsson et al, ). The third one is UroM medium, which is composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A‐DMEM) and MCDB 153 medium supplemented with 2.5% FBS or with physiological calcium concentration of 2.5 mM (Kreft et al, , ; Visnjar et al, ; Visnjar and Kreft, , ). Therefore, the comparison of the results and faster development of urothelial cultures suitable for tissue engineering are hindered.…”

Properties of the Urothelium that Establish the Blood–Urine Barrier and Their Implications for Drug Delivery

“…In addition to altered intracellular vesicle trafficking, endocytosed vesicles are capable of releasing their content, and the lipid membrane composition can affect the degree of leakage into the cytoplasm (Grasso and Calderon 2009). Superficial UC plasma membranes, with their urothelial plaques and rigidified lipids, should maximally prevent leakage of toxic urine substances into their cytoplasm, and apical endosomes do indeed exhibit very low (b) Expression and localization of UPs (anti-AUM, green) in porcine UCs in vitro cultured as is described in Višnjar and Kreft (2015). UPs (white arrowheads) are seen above the nuclei (blue).…”

“…Permeability can also be enhanced chemically with DMSO, protamine sulfate, hyaluronidase, or chitosan hydrochloride (GuhaSarkar and Banerjee 2010). In vitro and in vivo studies have indicated that chitosan hydrochloride is a promising agent in disrupting the urothelial barrier, which increases urothelial permeability in low concentrations and causes desquamation of superficial UCs in high concentrations (Erman et al 2013;Visnjar and Kreft 2014).…”

“…5 Glycocalyx of porcine urothelial cells (UCs) in vitro. (a) UCs in vitro, prepared as described in Višnjar and Kreft (2015), labeled with the lectin jacalin (green) and uroplakins (red, antibodies against uroplakins (anti-AUM)). Jacalin binds to O-glycosidically linked oligosaccharides, preferring the structure galactosyl (β-1,3) N-acetylgalactosamine.…”