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.
[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. http://dx.doi.org/10.1016/0968-0004(94)90170-810.1016/0968-0004(94)90170-8Search in 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. Search in Google Scholar
[3] Ferguson, D.R. Urothelial function. BJU Int. 84 (1999) 235–242. http://dx.doi.org/10.1046/j.1464-410x.1999.00187.x10.1046/j.1464-410x.1999.00187.xSearch in 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. http://dx.doi.org/10.1038/3503951910.1038/35039519Search in 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. http://dx.doi.org/10.1073/pnas.23124369810.1073/pnas.231243698Search in 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. http://dx.doi.org/10.1113/jphysiol.2004.07115910.1113/jphysiol.2004.071159Search in Google Scholar
[7] Szallasi, A. and Blumberg, P.M. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol. Rev. 51 (1999) 159–212. Search in 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. http://dx.doi.org/10.1290/1071-2690(2001)037<0419:EALTCO>2.0.CO;210.1290/1071-2690(2001)037<0419:EALTCO>2.0.CO;2Search in 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. Search in 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. http://dx.doi.org/10.1172/JCI11792810.1172/JCI117928Search in 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. Search in 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. http://dx.doi.org/10.1139/Y10-07210.1139/Y10-072Search in 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. Search in 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. 10.1152/ajprenal.1998.275.2.F226Search in Google Scholar
[15] de Groat, W.C. The urothelium in overactive bladder: passive bystander or active participant? Urology 64 (2004) 7–11. http://dx.doi.org/10.1016/j.urology.2004.08.06310.1016/j.urology.2004.08.063Search in 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. http://dx.doi.org/10.1016/S0022-5347(05)65726-610.1016/S0022-5347(05)65726-6Search in 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. http://dx.doi.org/10.1073/pnas.23124369810.1073/pnas.231243698Search in Google Scholar
[18] Lewczyński, D. and Ginalski, K. The interactome: predicting the proteinprotein interactions in cells. Cell. Mol. Biol. Lett. 14 (2009) 1–22. http://dx.doi.org/10.2478/s11658-008-0024-710.2478/s11658-008-0024-7Search in 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. http://dx.doi.org/10.2478/s11658-010-0015-310.2478/s11658-010-0015-3Search in 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. http://dx.doi.org/10.2478/s11658-010-0004-610.2478/s11658-010-0004-6Search in 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. Search in 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. Search in 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. http://dx.doi.org/10.1006/cbir.2000.065810.1006/cbir.2000.0658Search in 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. http://dx.doi.org/10.1016/S0074-7696(08)62312-810.1016/S0074-7696(08)62312-8Search in Google Scholar
[25] Duvall, E. and Wyllie, A.H. Death and the cell. Immunol. Today 7 (1986) 115–119. http://dx.doi.org/10.1016/0167-5699(86)90152-010.1016/0167-5699(86)90152-0Search in Google Scholar
[26] Arends, M.J. and Wyllie, A.H. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32 (1991) 223–254. 10.1016/B978-0-12-364932-4.50010-1Search in 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. http://dx.doi.org/10.1152/ajprenal.90718.200810.1152/ajprenal.90718.2008Search in Google Scholar PubMed PubMed Central
[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. Search in 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. Search in 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. Search in Google Scholar
[31] Geppetti, P., Nassini, R., Materazzi, S. and Benemei, S. The concept of neurogenic inflammation. BJU Int. 101Suppl. 3 (2008) 2–6. http://dx.doi.org/10.1111/j.1464-410X.2008.07493.x10.1111/j.1464-410X.2008.07493.xSearch in Google Scholar PubMed
[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. http://dx.doi.org/10.1055/s-0029-122460010.1055/s-0029-1224600Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1002/nau.2083010.1002/nau.20830Search in Google Scholar PubMed
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