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A non-enzymatic method for dissection of mouse bladder urothelial tissue

Nature Protocols volume 14, pages 1280–1292 (2019)Cite this article

Abstract

Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. In addition, PCR or immunoblot results obtained from homogenates of bladder mucosa or whole bladder do not represent pure urothelial cells. We describe a dissection process that does not require enzymes and is able to obtain pure urothelial tissues from mice and humans. This method can isolate single urothelial cells for electrophysiology in situ and can also isolate pure urothelial tissue for PCR, microarray, and immunoblot procedures. The time required to obtain urothelial tissue from one mouse bladder is 15–20 min. This method is simple and time efficient as compared with alternative methods and therefore facilitates our understanding of urothelial biology.

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Fig. 1: A cross-section of mouse bladder with H&E staining.
Fig. 2: Adjustment of the dissection forceps.
Fig. 3: Reagents and instruments for perfusion and operation should be positioned so they are all within easy reach during the dissection.
Fig. 4: Mucosa jutting from edge of a bladder piece.
Fig. 5: Dissection of different bladder compartments.
Fig. 6: Dissected urothelial tissue under a microscope.
Fig. 7: Removal of single cell from a sheet of urothelial tissue.
Fig. 8: Uroplakin subunit-Ib (UPIb) is expressed only in urothelial tissue.
Fig. 9: Microscope light sources for the dissection process.

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References

  1. Birder, L. & Andersson, K. E. Urothelial signaling. Physiol. Rev. 93, 653–680 (2013).

    Article  CAS  Google Scholar 

  2. Hamilton, C., Tan, L., Miethke, T. & Anand, P. K. Immunity to uropathogens: the emerging roles of inflammasomes. Nat. Rev. Urol. 14, 284–295 (2017).

    Article  CAS  Google Scholar 

  3. Chai, T. C., Russo, A., Yu, S. & Lu, M. Mucosal signaling in the bladder. Auton. Neurosci. 200, 49–56 (2016).

    Article  CAS  Google Scholar 

  4. Balsara, Z. R. & Li, X. Sleeping beauty: awakening urothelium from its slumber. Am. J. Physiol. Renal Physiol. 312, F732–F743 (2017).

    Article  CAS  Google Scholar 

  5. Watanabe, M. et al. Trpm7 protein contributes to intercellular junction formation in mouse urothelium. J. Biol. Chem. 290, 29882–29892 (2015).

    Article  CAS  Google Scholar 

  6. Miyamoto, T. et al. Functional role for Piezo1 in stretch-evoked Ca2+ influx and ATP release in urothelial cell cultures. J. Biol. Chem. 289, 16565–16575 (2014).

    Article  CAS  Google Scholar 

  7. Mochizuki, T. et al. The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J. Biol. Chem. 284, 21257–21264 (2009).

    Article  CAS  Google Scholar 

  8. Everaerts, W. et al. Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis. Proc. Natl. Acad. Sci. USA 107, 19084–19089 (2010).

    Article  CAS  Google Scholar 

  9. Kullmann, F. A. et al. Inflammation and tissue remodeling in the bladder and urethra in feline interstitial cystitis. Front. Syst. Neurosci. 12, 13 (2018).

    Article  Google Scholar 

  10. Beckel, J. M. et al. Pannexin 1 channels mediate the release of ATP into the lumen of the rat urinary bladder. J. Physiol. 593, 1857–1871 (2015).

    Article  CAS  Google Scholar 

  11. Yang, W., Searl, T. J., Yaggie, R., Schaeffer, A. J. & Klumpp, D. J. A MAPP network study: overexpression of tumor necrosis factor-α in mouse urothelium mimics interstitial cystitis. Am. J. Physiol. Renal Physiol. 315, F36–F44 (2018).

    Article  Google Scholar 

  12. Li, Y., Lu, M., Alvarez-Lugo, L., Chen, G. & Chai, T. C. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by female mouse bladder urothelial cells and expressed by the urothelium as an early response to lipopolysaccharides (LPS). Neurourol. Urodyn. 36, 1020–1025 (2017).

    Article  Google Scholar 

  13. Lu, M. et al. Lipopolysaccharide stimulates BK channel activity in bladder umbrella cells. Am. J. Physiol. Cell Physiol. 314, C643–C653 (2018).

    Article  CAS  Google Scholar 

  14. Acevedo-Alvarez, M. et al. Mouse urothelial genes associated with voiding behavior changes after ovariectomy and bladder lipopolysaccharide exposure. Neurourol. Urodyn. 37, 2398–2405 (2018).

    Article  CAS  Google Scholar 

  15. Niemczyk, G., Czarzasta, K., Radziszewski, P., Wlodarski, P. & Cudnoch-Jedrzejewska, A. Pathophysiological effect of bladder outlet obstruction on the urothelium. Ultrastruct. Pathol. 42, 317–322 (2018).

    Article  Google Scholar 

  16. Park, E. C. et al. Proteomic analysis of urothelium of rats with detrusor overactivity induced by bladder outlet obstruction. Mol. Cell. Proteomics 17, 948–960 (2018).

    Article  CAS  Google Scholar 

  17. Yang, S. W., Jeong, S. W. & Song, K. H. Increased expression of neuregulin 1 in the urothelium of rat bladder with partial bladder outlet obstruction. BMC Urol. 17, 115 (2017).

    Article  Google Scholar 

  18. Stenqvist, J., Carlsson, T., Winder, M. & Aronsson, P. Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder. Auton. Neurosci. 213, 60–70 (2018).

    Article  CAS  Google Scholar 

  19. Zhang, X. et al. Carbenoxolone inhibits TRPV4 channel-initiated oxidative urothelial injury and ameliorates cyclophosphamide-induced bladder dysfunction. J. Cell. Mol. Med. 21, 1791–1802 (2017).

    Article  CAS  Google Scholar 

  20. Lopez-Beltran, A., Montironi, R., Raspollini, M. R., Cheng, L. & Netto, G. J. Iatrogenic pathology of the urinary bladder. Semin. Diagn. Pathol. 35, 218–227 (2018).

    Article  Google Scholar 

  21. Doyle, C. et al. The role of the mucosa in modulation of evoked responses in the spinal cord injured rat bladder. Neurourol. Urodyn. 37, 1583–1593 (2018).

    Article  Google Scholar 

  22. Kullmann, F. A. et al. Urothelial proliferation and regeneration after spinal cord injury. Am. J. Physiol. Renal Physiol. 313, F85–F102 (2017).

    Article  CAS  Google Scholar 

  23. Ungerer, T. D. et al. Influence of urothelial or suburothelial cholinergic receptors on bladder reflexes in chronic spinal cord injured cats. Exp. Neurol. 285, 147–158 (2016).

    Article  CAS  Google Scholar 

  24. Ozbilgin, M. K. et al. Effects of cyclooxygenase on the urothelium of the urinary bladder of mice exposed to pelvic radiation. Anal. Quant. Cytopathol. Histopathol. 38, 103–110 (2016).

    Google Scholar 

  25. Kanai, A. J. et al. Manganese superoxide dismutase gene therapy protects against irradiation-induced cystitis. Am. J. Physiol. Renal Physiol. 283, F1304–F1312 (2002).

    Article  CAS  Google Scholar 

  26. Chang, A. et al. Social stress in mice induces voiding dysfunction and bladder wall remodeling. Am. J. Physiol. Renal Physiol. 297, F1101–F1108 (2009).

    Article  CAS  Google Scholar 

  27. Hawthorn, M. H., Chapple, C. R., Cock, M. & Chess-Williams, R. Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br. J. Pharmacol. 129, 416–419 (2000).

    Article  CAS  Google Scholar 

  28. Andersson, M., Aronsson, P., Doufish, D., Lampert, A. & Tobin, G. Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder. Auton. Neurosci. 170, 5–11 (2012).

    Article  CAS  Google Scholar 

  29. Chaiyaprasithi, B., Mang, C. F., Kilbinger, H. & Hohenfellner, M. Inhibition of human detrusor contraction by a urothelium derived factor. J. Urol. 170, 1897–1900 (2003).

    Article  CAS  Google Scholar 

  30. Saban, M. R., Nguyen, N. B., Hammond, T. G. & Saban, R. Gene expression profiling of mouse bladder inflammatory responses to LPS, substance P, and antigen-stimulation. Am. J. Pathol. 160, 2095–2110 (2002).

    Article  CAS  Google Scholar 

  31. Li, M., Sun, Y., Simard, J. M., Wang, J. Y. & Chai, T. C. Augmented bladder urothelial polyamine signaling and block of BK channel in the pathophysiology of overactive bladder syndrome. Am. J. Physiol. Cell Physiol. 297, C1445–C1451 (2009).

    Article  CAS  Google Scholar 

  32. National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals. in Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington, DC, 2011).

  33. Deng, F. M. et al. Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly. J. Cell Biol. 159, 685–694 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank L. Sanders for editing the manuscript, L. Alvarez-Lugo for RT–PCR assistance, J. Lu for narration of the video, and K. Dong for help in taking H&E images. This paper was supported by Yale University institutional funding.

Author information

Authors and Affiliations

  1. Department of Urology, Yale University School of Medicine, New Haven, CT, USA

    Ming Lu, Peter G. Schulam & Toby C. Chai

  2. Department of Urology, Qilu Hospital of Shandong University, Jinan, China

    Kejia Zhu & Toby C. Chai

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Contributions

M.L. and T.C.C. conceived and designed the research. M.L. demonstrated the dissection and edited the video. M.L. and K.Z. performed the experiments. M.L. and T.C.C. drafted the manuscript. M.L., P.G.S., and T.C.C. edited and revised the manuscript. All the authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Ming Lu or Toby C. Chai.

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The authors declare no competing interests.

Additional information

Journal peer review information: Nature Protocols thanks David Klumpp and Hiroshi Miyamoto for their contribution to the peer review of this work.

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Related links

Key reference(s) using this protocol

Li, Y., Lu, M., Alvarez-Lugo, L., Chen, G. & Chai, T. C. Neurourol. Urodyn. 36, 1020–1025 (2017): https://doi.org/10.1002/nau.23057

Lu, M. et al. Am. J. Physiol. Cell Physiol. 314, C643–C653 (2018): https://doi.org/10.1152/ajpcell.00339.2017

Acevedo-Alvarez, M. et al. Neurourol. Urodyn. 37, 2398–2405 (2018): https://doi.org/10.1002/nau.23592

Integrated supplementary information

Supplementary Figure 1 Dissected sheet of human urothelial tissue under a microscope.

A, B and C are the same dissected sheet of urothelial tissue from a human bladder at different magnifications. The basal cells are shown facing up. The underlining intermediate cells and umbrella cells could not be seen without removal of the basal cells. Scale bars in: A = 400 µm; B = 200 µm; C = 100 µm. (Human bladder obtained from cystectomy specimens. The Yale IRB approved the protocol.).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1

Reporting Summary

Supplementary Video 1

How to dissect urothelial tissue from mouse bladder. Part 1: making the dissection ice dish. Supplementary Video 1 © The Author(s), under license to Springer Nature America, Inc. 2019.

Supplementary Video 2

How to dissect urothelial tissue from mouse bladder. Part 2: perfusing the mouse with PBS. Supplementary Video 2 © The Author(s), under license to Springer Nature America, Inc. 2019.

Supplementary Video 3

How to dissect urothelial tissue from mouse bladder. Part 3: dissecting urothelial tissue. Supplementary Video 3 © The Author(s), under license to Springer Nature America, Inc. 2019.

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Lu, M., Zhu, K., Schulam, P.G. et al. A non-enzymatic method for dissection of mouse bladder urothelial tissue. Nat Protoc 14, 1280–1292 (2019). https://doi.org/10.1038/s41596-019-0142-x

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