Skip to main content
  • Short Communication
  • Published:

The effect of hyperosmolar stimuli and cyclophosphamide on the culture of normal rat urothelial cells in vitro

Abstract

Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells (RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively. The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively, 14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%, respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l HS had the most harmful effect based on the percentage of necrotic and apoptotic cells.

Abbreviations

CYP:

cyclophosphamide

CGRP:

calcitonin gene-related peptide

DO:

detrusor overactivity

FBS:

fetal bovine serum

GAG:

glycosaminoglycans

HEPES:

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

HS:

hyperosmolar stimuli

LUTS:

lower urinary tract symptoms

MEM:

minimal essential medium

RUCC:

rat urothelial cultured cells

TRPV1:

transient receptor potential vanilloid subtype 1

TRPV4:

transient receptor potential vanilloid subtype 4

TRPM8:

transient receptor potential vanilloid subfamily M member 8

TRPA1:

transient receptor potential ankyrin subtype 4

SP:

substance P

References

  1. Martin, S.J., Green, D.R. and Cotter, T.G. Dicing with death: dissecting the components of the apoptosis machinery. Trends. Biochem. Sci. 19 (1994) 26–30.

    Article  PubMed  CAS  Google Scholar 

  2. Alberts, K., Johnson, A., Lewis, J., Raff, M. and Roberts, W.P. Programmed cell death eliminates unwanted cells in: Molecular Biology of the Cell (textbook) 5th ed., 2008, Garland Science p. 1115.

  3. Ferguson, D.R. Urothelial function. BJU Int. 84 (1999) 235–242.

    Article  PubMed  CAS  Google Scholar 

  4. Cockayne, D.A., Hamilton, S.G., Zhu, Q.M., Dunn, P.M., Zhong, Y., Novakovic, S., Malmberg, A.B., Cain, G., Berson, A., Kassotakis, L., Hedley, L., Lachnit, W.G., Burnstock, G., McMahon, S.B. and Ford, A.P. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 407 (2000) 1011–1015.

    Article  PubMed  CAS  Google Scholar 

  5. Birder, L.A., Kanai, A.J., de Groat, W.C., Kiss, S., Nealen, M.L., Burke, N.E., Dineley, K.E., Watkins, S., Reynolds, I.J. and Caterina, M.J. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc. Natl. Acad. Sci. USA 98 (2001) 13396–13401.

    Article  PubMed  CAS  Google Scholar 

  6. Chopra, B., Barrick, S.R., Meyers, S., Beckel, J.M., Zeidel, M.L., Ford, A.P.D.W., de Groat, W.C. and Birder, L.A. Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium. J. Physiol. 562 (2005) 859–871.

    Article  PubMed  CAS  Google Scholar 

  7. Szallasi, A. and Blumberg, P.M. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol. Rev. 51 (1999) 159–212.

    PubMed  CAS  Google Scholar 

  8. Zhang, Y.Y., Ludwikowski, B., Hurst, R. and Frey, P. Expansion and longterm culture of differentiated normal rat urothelial cells in vitro. In Vitro Cell. Dev. Biol. Anim. 37 (2001) 419–429.

    Article  PubMed  CAS  Google Scholar 

  9. Noguchi, S., Yura, Y., Sherwood, E.R., Kakinuma, H., Kashihara, N. and Oyasu, R. Stimulation of stromal cell growth by normal rat urothelial cell derived epidermal growth factor. Lab. Invest. 62 (1990) 538–544.

    PubMed  CAS  Google Scholar 

  10. Garland, A., Jordan, J.E., Necheles, J., Alger, L.E., Scully, M.M., Miller, R.J., Ray, D.W., White, S.R. and Solway, J. Hypertonicity, but not hypothermia, elicits substance P release from rat C-fiber neurons in primary culture. J. Clin. Invest. 95 (1995) 2359–2366.

    Article  PubMed  CAS  Google Scholar 

  11. Juszczak, K., Krolczyk, G., Filipek, M., Dobrowolski, Z.F. and Thor, P.J. Animal models of overactive bladder: cyclophosphamide (CYP)-induced cystitis in rats. Fol. Med. Cracov 48 (2007) 113–123.

    CAS  Google Scholar 

  12. Juszczak, K., Ziomber, A., Wyczółkowski, M. and Thor, P.J. Hyperosmolarity alters the micturition: The comparison of urinary bladder motor activity in hyperosmolar and cyclophosphamide-induced models of overactive bladder. Can. J. Physiol. Pharmacol. 88 (2010) 899–906.

    Article  PubMed  CAS  Google Scholar 

  13. Kulick, L.J., Clemons, D.J., Hall, R.L. and Koch, M.A. Refinement of the urine concentration test in rats. Contemp. Top. Lab. Anim. Sci. 44 (2005) 46–49.

    PubMed  CAS  Google Scholar 

  14. Birder, L.A., Apodaca, G., de Groat, W.C. and Kanai, A.J. Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am. J. Physiol. Renal. Physiol. 275 (1998) F226–F229.

    CAS  Google Scholar 

  15. de Groat, W.C. The urothelium in overactive bladder: passive bystander or active participant? Urology 64 (2004) 7–11.

    Article  PubMed  Google Scholar 

  16. Sun, Y., Keay, S., DeDeyne, P. and Chai, T.C. Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis. J. Urol. 166 (2001) 1951–1956.

    Article  PubMed  CAS  Google Scholar 

  17. Birder, L.A., Kanai, A.J., de Groat, W.C., Kiss, S., Nealen, M.L., Burke, N.E., Dineley, K.E., Watkins, S., Reynolds, I.J. and Caterina, M.J. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc. Natl. Acad. Sci. USA 98 (2001) 13396–13401.

    Article  PubMed  CAS  Google Scholar 

  18. Lewczyński, D. and Ginalski, K. The interactome: predicting the proteinprotein interactions in cells. Cell. Mol. Biol. Lett. 14 (2009) 1–22.

    Article  Google Scholar 

  19. Rolius, R., Antoniou, C., Nazarova, L.A., Kim, S.H., Cobb, G., Gala, P., Rajaram, P., Li, Q. and Fung, L.W.M. Inhibition of calpain but not caspase activity by spectrin fragments. Cell. Mol. Biol. Lett. 15 (2010) 395–405.

    Article  PubMed  CAS  Google Scholar 

  20. Urbanova, M., Plzak, J., Strnad, H. and Betka, J. Circulating nucleic acids as a new diagnostic tool. Cell. Mol. Biol. Lett. 15 (2010) 242–259.

    Article  PubMed  CAS  Google Scholar 

  21. Philips, F.S., Sternberg, S.S., Cronin, A.P. and Vidal, P.M. Cyclophosphamide and urinary bladder toxicity. Cancer Res. 21 (1961) 1577–1589.

    PubMed  CAS  Google Scholar 

  22. Locher, G.W. and Cooper, E.H. Repair of rat urinary bladder epithelium following injury by cyclophosphamide. Invest. Urol. 8 (1970) 116–123.

    PubMed  CAS  Google Scholar 

  23. Romih, R., Koprivec, D., Martinic, D.S. and Jezernik, K. Restoration of the rat urothelium after cyclophosphamide treatment. Cell. Biol. Int. 25 (2001) 531–537.

    Article  PubMed  CAS  Google Scholar 

  24. Wyllie, A.H., Kerr, J.F.R. and Currie, A.R. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68 (1980) 251–306.

    Article  PubMed  CAS  Google Scholar 

  25. Duvall, E. and Wyllie, A.H. Death and the cell. Immunol. Today 7 (1986) 115–119.

    Article  CAS  Google Scholar 

  26. Arends, M.J. and Wyllie, A.H. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32 (1991) 223–254.

    PubMed  CAS  Google Scholar 

  27. Kullmann, F.A., Shah, M.A., Birder, L.A. and de Groat, W.C. Functional TRP and ASIC-like channels in cultured urothelial cells from the rat. Am. J. Physiol. Renal. Physiol. 296 (2009) F892–F901.

    Article  PubMed  CAS  Google Scholar 

  28. Juszczak, K., Ziomber, A., Wyczółkowski, M. and Thor, P.J. Urodynamic effects of the bladder C-fiber afferent activity modulation in chronic overactive bladder model rats. J. Physiol. Pharmacol. 60 (2009) 85–91.

    PubMed  CAS  Google Scholar 

  29. Juszczak, K., Gil, K., Wyczółkowski, M. and Thor, P.J. Functional, histological structure and mastocytes numbers alterations in rat urinary bladders following acute and chronic cyclophosphamide treatment. J. Physiol. Pharmacol. 61 (2010) 477–482.

    PubMed  CAS  Google Scholar 

  30. Juszczak, K., Wyczółkowski, M. and Thor, P.J. Urodynamic evaluation of experimental rat models of urinary bladder dysfunction: a systematic review of the literature. Ann. Urol. 1 (2011) 1–13.

    Google Scholar 

  31. Geppetti, P., Nassini, R., Materazzi, S. and Benemei, S. The concept of neurogenic inflammation. BJU Int. 101Suppl. 3 (2008) 2–6.

    Article  PubMed  CAS  Google Scholar 

  32. Gauruder-Burmester, A. and Popken, G. Follow-up at 24 months after treatment of overactive bladder with 0.2% sodium chondroitin sulphate. Aktuelle Urol. 40 (2009) 355–359.

    Article  PubMed  Google Scholar 

  33. Yeh, C.H., Chiang, H.S. and Chien, C.T. Hyaluronic acid ameliorates bladder hyperactivity via the inhibition of H2O2-enhanced purinergic and muscarinic signalling in the rat. Neurourol. Urodyn. 29 (2010) 765–770.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kajetan Juszczak.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Juszczak, K., Kaszuba-Zwoińska, J., Chorobik, P. et al. The effect of hyperosmolar stimuli and cyclophosphamide on the culture of normal rat urothelial cells in vitro . Cell Mol Biol Lett 17, 196–205 (2012). https://doi.org/10.2478/s11658-012-0002-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-012-0002-y

Key words