Histochemistry and Cell Biology

, Volume 131, Issue 1, pp 129–139 | Cite as

Rapid differentiation of superficial urothelial cells after chitosan-induced desquamation

  • Peter Veranič
  • Andreja Erman
  • Mojca Kerec-Kos
  • Marija Bogataj
  • Aleš Mrhar
  • Kristijan Jezernik
Original Paper


Superficial cell desquamation followed by differentiation of newly exposed superficial cells induces regeneration of the urinary bladder epithelium, urothelium. In the present work, chitosan was evaluated as a new inducer of urothelial cell desquamation, in order to study the regeneration of mouse urothelial cells in vivo. Intravesical application of chitosan dispersion caused complete removal of only the superficial layer of cells within 20 min of treatment. Differentiation of the new superficial layer was followed by the appearance and distribution of three urothelial differentiation markers, tight junction protein ZO1, cytokeratin 20 and the maturation of the apical plasma membrane. The arrangement of ZO1 into continuous lines in individual cells of the intermediate layer was already found after 10 min of chitosan application, when desquamation had just started. The appearance of the apical membrane changed from microvillar to typically scalloped within 20 min of regeneration, while complete arrangement of the cytokeratin 20 network took 60 min. These findings provide a new perspective on the rate of the differentiation process in the urothelium and make chitosan a new and a very controllable tool for studies on urothelial regeneration.


Chitosan Desquamation Urinary bladder Regeneration Differentiation 


  1. Acharya P, Beckel J, Ruiz WG, Wang E, Rojas R, Birder L, Apodaca G (2004) Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium. Am J Physiol Renal Physiol 287:F305–F318PubMedCrossRefGoogle Scholar
  2. Aronson M, Medalia O, Amichay D, Nativ O (1988) Endotoxin-induced shedding of viable uroepithelial cells is an antimicrobial defense mechanism. Infect Immun 56:1615–1617PubMedGoogle Scholar
  3. Artursson P, Lindmark T, Davis SS, Illum L (1994) Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm Res 11:1358–1361PubMedCrossRefGoogle Scholar
  4. Baskin L, Meaney D, Landsman A, Zderic SA, Macarak E (1994) Bovine bladder compliance increases with normal fetal development. J Urol 152:692–695PubMedGoogle Scholar
  5. Baskin LS, Hayward SW, Young PF, Cunha GR (1996) Ontogeny of the rat bladder: smooth muscle and epithelial differentiation. Acta Anat (Basel) 155:163–171CrossRefGoogle Scholar
  6. Dalal E, Medalia O, Harari O, Aronson M (1994) Moderate stress protects female mice against bacterial infection of the bladder by eliciting uroepithelial shedding. Infect Immun 62:5505–5510PubMedGoogle Scholar
  7. Erman A, Jezernik K, Stiblar-Martincic D, Romih R, Veranic P (2001) Postnatal restoration of the mouse urinary bladder urothelium. Histochem Cell Biol 115:309–316PubMedGoogle Scholar
  8. Farsund T (1975) Cell kinetics of mouse urinary bladder epithelium. I. Circadian and age variations in cell proliferation and nuclear DNA content. Virchows Arch B Cell Pathol 18:35–49PubMedGoogle Scholar
  9. Goldstein P, Kroemer G (2006) Cell death by necrosis: towards a molecular definition. TRENDS Biochem Sci 32:37–43CrossRefGoogle Scholar
  10. Hasegawa M, Yagi K, Iwakawa S, Hirai M (2001) Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells. Jpn J Cancer Res 92:459–466PubMedGoogle Scholar
  11. Hejazi R, Amiji M (2003) Chitosan-based gastrointestinal delivery systems. J Control Release 89:151–165PubMedCrossRefGoogle Scholar
  12. Hu P, Meyers S, Liang FX, Deng FM, Kachar B, Zeidel ML, Sun TT (2002) Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability. Am J Physiol Renal Physiol 283:F1200–F1207PubMedGoogle Scholar
  13. Jacob J, Hindmarsh JR, Ludgate CM, Chisholm GD (1982) Observations on the ultrastructure of human urothelium: the response of normal bladder of elderly subjects to hyperthermia. Urol Res 10:227–237PubMedCrossRefGoogle Scholar
  14. Jenings CD, Boleyn K, Bridges SR, Wood PJ, Anderson JW (1988) A comparison of the lipid-lowering and intestinal morphological effects of cholestyramine, chitosan, and oat gum in rats. Proc Soc Exp Biol Med 189:13–20Google Scholar
  15. Jost SP (1989) Cell cycle of normal bladder urothelium in developing and adult mice. Virchows Arch B Cell Pathol Incl Mol Pathol 57:27–36PubMedCrossRefGoogle Scholar
  16. Kachar B, Liang F, Lins U, Ding M, Wu XR, Stoffler D, Aebi U, Sun TT (1999) Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J Mol Biol 15:595–608CrossRefGoogle Scholar
  17. Kerec M, Bogataj M, Veranic P, Mrhar A (2005) Permeability of pig urinary bladder wall: the effect of chitosan and the role of calcium. Eur J Pharm Sci 25:113–121PubMedCrossRefGoogle Scholar
  18. Kos MK, Bogataj M, Veranic P, Mrhar A (2006) Permeability of pig urinary bladder wall: time and concentration dependent effect of chitosan. Biol Pharm Bull 29:1685–1691PubMedCrossRefGoogle Scholar
  19. Koss LG (1967) A light and electron microscopic study of the effects of a single dose of cyclophosphamide on various organs in the rat. I. The urinary bladder. Lab Invest 16:44–65PubMedGoogle Scholar
  20. Kotzé AF, Luessen HL, de Leeuw BJ, de Boer BG, Verhoef JC, Junginger HE (1997) N-Trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2). Pharm Res 14:1197–1202PubMedCrossRefGoogle Scholar
  21. Kreft ME, Sterle M, Veranic P, Jezernik K (2005) Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation. Histochem Cell Biol 123:529–539PubMedCrossRefGoogle Scholar
  22. Kunze E, Wöltjen HH, Nehm FJ, Schauer A (1981) Cell cycle kinetics of regenerating urothelial cells of the rat urinary bladder after partial cystectomy. Virchows Arch B Cell Pathol Incl Mol Pathol 38:117–125PubMedCrossRefGoogle Scholar
  23. Levi PE, Cooper EH, Anderson CK, Williams RE (1969) Analyses of DNA content, nuclear size and cell proliferation of transitional cell carcinoma in man. Cancer 23:1074–1085PubMedCrossRefGoogle Scholar
  24. Locher GW, Cooper EH (1970) Repair of rat urinary bladder epithelium following injury by cyclophosphamide. Invest Urol 8:116–123PubMedGoogle Scholar
  25. Luna EJ, Hitt AL (1992) Cytoskeleton-plasma membrane interactions. Science 258:955–964 ReviewPubMedCrossRefGoogle Scholar
  26. Messier B, Leblond CP (1960) Cell proliferation and migration as revealed by adioautography after injection of thymidine-H3 into male rats and mice. Am J Anat 106:247–285PubMedCrossRefGoogle Scholar
  27. Miura T, Usami M, Tsuura Y, Ishida H, Seino Y (1995) Hypoglycemic and hypolipidemic effect of chitosan in normal and neonatal streptozotocin-induced diabetic mice. Biol Pharm Bull 18:1623–1625PubMedGoogle Scholar
  28. Moll R, Wu XR, Lin JH, Sun TT (1995) Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas. Am J Pathol 147:1383–1397PubMedGoogle Scholar
  29. Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J, Hultgren SJ (1998) Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 282:1494–1497PubMedCrossRefGoogle Scholar
  30. Mulvey MA, Schilling JD, Hultgren SJ (2001) Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection. Infect Immun 69:4572–4579PubMedCrossRefGoogle Scholar
  31. Nativ O, Dalal E, Hidas G, Aronson M (2006) EDTA-induced urothelial cell shedding for the treatment of superficial bladder cancer in the mouse. Int J Urol 13:1344–1346PubMedCrossRefGoogle Scholar
  32. Philips FS, Sternberg SS, Cronin AP, Vidal PM (1961) Cyclophosphamide and urinary bladder toxicity. Cancer Res 21:1577–1589PubMedGoogle Scholar
  33. Ranaldi G, Marigliano I, Vespignani I, Perozzi G, Sambuy Y (2002) The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J Nutr Biochem 13:157–167PubMedCrossRefGoogle Scholar
  34. Romih R, Jezernik K, Masera A (1998) Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment. Histochem Cell Biol 109:263–269PubMedCrossRefGoogle Scholar
  35. Romih R, Veranic P, Jezernik K (1999) Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder. Histochem Cell Biol 112:375–380PubMedCrossRefGoogle Scholar
  36. Romih R, Koprivec D, Martincic DS, Jezernik K (2001) Restoration of the rat urothelium after cyclophosphamide treatment. Cell Biol Int 25:531–537PubMedCrossRefGoogle Scholar
  37. Şenel S, Hincal AA (2001) Drug permeation enhancement via buccal route: possibilities and limitations. J Control Release 72:133–144PubMedCrossRefGoogle Scholar
  38. Stewart FA (1986) Mechanism of bladder damage and repair after treatment with radiation and cytostatic drugs. Br J Cancer Suppl 7:280–291PubMedGoogle Scholar
  39. Truschel ST, Wang E, Ruiz WG, Leung SM, Rojas R, Lavelle J, Zeidel M, Stoffer D, Apodaca G (2002) Stretch-regulated exocytosis/endocytosis in bladder umbrella cells. Mol Biol Cell 13:830–846PubMedCrossRefGoogle Scholar
  40. Veranic P, Jezernik K (2000) The response of junctional complexes to induced desquamation in mouse bladder urothelium. Biol Cell 92:105–113PubMedCrossRefGoogle Scholar
  41. Veranic P, Jezernik K (2002) Trajectorial organisation of cytokeratins within the subapical region of umbrella cells. Cell Motil Cytoskeleton 53:317–325PubMedCrossRefGoogle Scholar
  42. Veranic P, Romih R, Jezernik K (2004) What determines differentiation of urothelial umbrella cells? Eur J Cell Biol 83:27–34PubMedCrossRefGoogle Scholar
  43. Wang Y, Chen M, Wolosin JM (1993) ZO-1 in corneal epithelium; stratal distribution and synthesis induction by outer cell removal. Exp Eye Res 57:283–292PubMedCrossRefGoogle Scholar
  44. Watanabe S, Sasaki J (1992) 12-O-Tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium. Cell Tissue Res 268:239–245PubMedCrossRefGoogle Scholar
  45. Wolosin JM, Chen M (1993) Ontogeny of corneal epithelial tight junctions: stratal locale of biosynthetic activities. Invest Ophthalmol Vis Sci 34:2655–2664PubMedGoogle Scholar
  46. Yu S, Zhao Y, Wu F, Zhang X, Lu W, Zhang H, Zhang Q (2004) Nasal insulin delivery in the chitosan solution: in vitro and in vivo studies. Int J Pharm 281:11–23PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Peter Veranič
    • 1
  • Andreja Erman
    • 1
  • Mojca Kerec-Kos
    • 2
  • Marija Bogataj
    • 2
  • Aleš Mrhar
    • 2
  • Kristijan Jezernik
    • 1
  1. 1.Institute of Cell Biology, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Faculty of PharmacyUniversity of LjubljanaLjubljanaSlovenia

Personalised recommendations