Novel Roles of TRP Channel in Urinary Bladder

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Research Conducted on this Topic in China

The research conducted on the role of TRP and TRP superfamily in China is still scarce and
limited in scope. However, progress has been made in recent years with several universities and
medical institutes beginning to show interest in the topic. In 2017, Du YU, Shi Ying Jin, Zhang Zan
Hui, Jia Ni-ya, and Fan Jun-Ying, all of the hospital of Baotou Medical College of inner Mongolia,
China, conducted a study on the expression and significance of TRPC6 and a-acticin-4 in renal tissue
of rats with Adriamycin nephropathy. Their study focused on the different gene expressions between
of TRPC6 in different kidney tissues to establish the interaction and co-interaction of TRP molecules
and of nephrotic-proteinuria. They concluded that increased expression of TRP6 resulted in increased
damage of foot cells and worsening of glomerulosclerosis. This research followed the 2013 research
on relationship between TRP ion channel family and bladder function, by Li Chuangzhang of
Tengzhou Centra People’s Hospital, Tengzhou, Shandong, China. In his research paper, Li
Chuangzhang noted the limitation of research in the area of TRP roles in the bladder in China. There
has also been research in related areas of study but not specifically on the topic proposed. Jiang
Wang, Guojun Chen, Xing Zhao and myself conducted a research on microRNA-204 modulates and
their importance in malignant transformation and its dysregulation in prostate cancer. In our
conclusion we noted the downgrading of miR-204 in chemo resistant PC samples and cells. While
they are evidence of significant progress in Chinese research in TRP and their specific role in
different organs, there is still room for further research in the topic.

Thus although there has been research in TRP in general, more specialized research is
required to comprehensively understand the role of TRP in the bladder and consequently its role in
physiological and pathophysiological conditions such as prostate cancer.

Research Conducted on this Topic Abroad

Unlike in China, extensive research has been conducted elsewhere on Transient Receptor
Potential particularly their role in the urinary bladder and tract. In 2010 Karl-Erik Anderson,
Christian Gratzke, and Petter Hedlund, of Institute of Regenerative Medicine, USA, conducted
research on the role of the transient receptor potential superfamily of cation-selective channels in the
management of overactive bladder. Their findings confirmed the link between the activation of
members of the TRP superfamily and the lower urinary tracts (LUTS) and called for further
investigation on the novel purpose of TRP superfamily in LUT function. Further research by Kiril l.
Hristov, Amy Smith, Shankar parajuli, John Malyz, Eric Rovner, and Georgi Petkov, in 2016, set out
to establish the novel regulatory mechanism in human urinary bladder. They established, in this
study, that unlike in guinea pigs and rats, TRPM4 channel had different effect on human DSM. This
and other research projects, such as those by Kirk Hamilton, have set the stage for further study into
the investigation of potential therapeutic targets for genetic treatment of Over-Active-Bladder
syndrome.
 Novel Roles of TRP Channel in Urinary Bladder 3

Since TRPs identification and cloning in 1989 by Craig Montell and Gerald Rubin, extensive
research has been conducted on TRP in the USA and Europe. However, further research is required
to establish the novel role of different TRP superfamily members in different organs e.g. the bladder.
Furthermore, a research collaboration among all these regions will widen the knowledge pool on the
subject and give relevant research a global perspective. This would fix the knowledge gap between
these regions and prevent repetitive research thus increasing resources available for new and
advanced research into specific areas of subject.

Academic Level and Advantages of Studying at Charite University, Berlin, Germany

Ranked 55th globally in clinical, pre-clinical and health, Charite University offers state of the art
facilities for medical research at any level, particularly CSC research. Hosting one of the largest
university hospitals in the world, with over 300 years in operation, the University offers modern
laboratory facilities that will offer me an unprecedented opportunity to conduct substantive research.
The new state-of-the-art innovative biobank, opened in 2016 will be an ideal environment to conduct
detailed experiments on different TRP samples and CSC. The opportunity to conduct CSC research
at Charite University is advantageous because of the following reasons:

1. Having already participated in two national nature science foundation of China (NSFC)
experiments resulting in increased experimental skills, conducting research in a European
University of Charite’s caliber will offer me much required broadening of my international
vision.
2. The large pool of specialists conducting Cancer Stem Cell research at Charite -
Universitätsmedizin Berlin, will offer me the appropriate environment and challenge to learn
and benefit from these researchers and specialists. I am particularly interested in the recent
discovery of cancer stem cells dependency on the ‘Hedgehog signaling pathway’ conducted
by Regan et al. in 2017 and published in the Cell Reports journal (Issue 10, Vol. 21).
3. Gain strict scientific guidance. My research supervisor, Prof. Mail Gollasch is the research
group leader at the Experimental and Clinical Research at Charite and Max Delbrück Center
of Molecular Medicine in the Helmholtz Association in Berlin, Germany. His research
focuses on nephrology and nephrogenetics. He has also conducted research on CSC and has
written over 70 journal articles different journals including, nature, Arteriosclerosis –
Thrombosis – and Vascular Biology, Phsiologica, and Current Hypertension Reports.
4. Conducting my research in a reputable institution such as Charite will not only allow me the
opportunity to explore and understand the German medical practice but will also offer me the
opportunity to take advantage of expanding my scope of understanding of CSC and TRP from
current ongoing projects at the University. Some of these current projects that my research
will draw from include; TMEM16a and TRP channels projects – the efforts of this project to
discover the novel mechanisms underlying TRPC6 and TRPV4 related to kidney pathologies
will be greatly useful to my research and the findings will be reviewed extensively.
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The Feasibility of Studying Abroad

Having participated in two extensive ‘Cancer Stem Cell’ researches granted by national
nature science foundation of China, I have gained a deep understanding of experimental skills and
the Chinese medical research environment. My participation in the research on the correlation
between MicroRNA-204 and prostate cancer was a great primary experience in the preparation for
this research. I gained very specific skills working on this project and understood some of the very
primary aspects of research methods and experimental practice. Moreover, I have professional skills
in separation and passivation of nucleic acids: gene knockout and knock on; miRNA microarray
assay; Protein (enzyme) extraction, isolation, passivation, identification; Cell culture; Plasmid
construction; Luciferase assays; Cell viability assay; Cell chemo sensitivity assay; Cell apoptosis
assay; Cell growth and detection of cell cycle; Detection of cell aging; Western blotting. The
facilities, knowledge pool, and opportunities offered by the University will allow me to put these
skills to practical use while learning international approaches to CSC research.

My unswerving interest in urology and Cancer Stem Cell requires the international exposure
necessary to widen my scope of understanding of both areas. My completion of the project research,
on the correlation between microRNA and a progression of prostate cancer and guiding clinical
treatment, at Chinese Academy of Medical Sciences gave me the necessary theoretical background
for theoretical learning and the ability to practically implement such theories in real world problems.
Further, my publication of microRNA-204 modulates, chemo-sensitivity and apoptosis of apoptosis
of prostate cancer cells by targeting Zinc-finger E-Box-Binding home box 1 as the first author in
American Journal of Transnational research is evidence of my commitment to the field of CSC
research and international corroboration to produce useful research on the subject.

I will bring to the institution, particularly my research group, a cheerful personality and
considerable knowledge in CSC research and comprehensive plan making and designation skills
learned from a work experience, research, and different academic programs. Indeed, my research on
microRNA-204 in 2017 at the Department of Urology, Qinghai University Affiliated Hospital, not
only improved my research skills but acts as evidence of my ability to lead a research team and
ensure research goals are achieved conclusively. Consequently, the publication of our findings in the
research is evidence of my ability to write scientific literature.

Having worked, studied, and conducted research in China, studying abroad, especially in an
internationally renowned institution like Charite, in a globally leading country in science, Germany,
is both timely and profound. The opportunity to work under the supervision of a world renowned
supervisor, Prof. Gollasch, in one of Europe’s largest medical research universities and post-modern
laboratory will offer me the opportunity to widen my scientific outlook and increase my knowledge
in the proposed topic significantly.
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Purpose and Expected Objectives of the Research Project

The purpose of this research is to establish the novel role of transient receptor potential (TRP)
in the bladder. The presence of TRP in both urothelium and detrusor muscle makes it a significant
player in the functions of the bladder as noted by Magdalene Moran et al. in Nature Reviews Drug
Discovery journal of August 2011. Hitherto, a major part of research has gone into TRP superfamily
in the kidney and the therapeutic potential of TRPs, for instance the possible use of TRP8 in
alleviating immunosuppression and as therapy in painful bladder disorders.

The expected objectives of the research will be to establish the benefits and detriments of
activation or inhibition of different TRPs in the bladder. While extensive research has shown the
benefits of TRPs in organs like the kidney, these results cannot be generalized. Yosuke Kaneko and
Arpad Szallasi take note of this in their article titled, Transient Receptor Potential Channels: A
Clinical Perspective, published in 2014 on the British Journal of Pharmacology. They note that
because of their irregular homology, members of the TRP superfamily, the benefits of one type of
TRP channel in one organ might be detrimental to another. Without the possibility of generalization,
it is important that their role in the bladder is given special attention. Indeed, as stated by Kirk
Hamilton, as the population’s life expectancy increases, a comprehensive understanding of TRP role
in the bladder might offer new approaches to therapeutic treatment of over-active bladder syndrome
(OAB) and prostate cancer.

Study specific Implementation Methods

Phase 2: Formulate Phase 3: Modify

Hypothesis original hypothesis

Phase 1: Observe Phase 3: Collect Phase 4: Establish

samples samples to test theory based on
hypothesis repeated vallidaton
of results

Figure 1 Primary research flow chart

The findings of this research are expected to bring to the fore the importance of transient
receptor potential, their role in the bladder, and how specific members of TRP superfamily can be
utilized for specialized treatment of urinary bladder diseases. Thus this research lies in the interface
of several medical fields; drug discovery, clinical health, and neurology. Through the research
process all through to implementation of results the following methodology will be implemented
 Novel Roles of TRP Channel in Urinary Bladder 6

Methods

Collection of Human DSM tissue

The Charite University hospital theatre will be used to collect DSM tissue from donor patients.
Currently available DSM tissue at Charite laboratory will also be used in cases of lack of donors as
well as for comparison purposes. All procedure for collection of human tissue will be approved by
Charite University Hospital management and my research supervisor, Prof. Maik Gollasch. After the
collection and preservation of human tissue, DSM single cell will be isolated using density gradient
centrifugation or any other cell-isolation technology suggested by supervisor and or research group.

RNA Extraction

RNA extraction and sequencing will then be conducted using the RNeasy Mini Kit. Extracted RNA
will then be reverse-transcribed into cDNA using virus reverse transcriptase and oligo primers.
Further, western blotting experiment will be conducted on DSM tissues by using precast SDS-PAGE
and transfers to immobillon-P membrane.

Immunohistochemistry, Immunocytochemistry, Data and Statistic Analysis

Immunohistochemistry and immunocytochemistry will then be done with isolated DSM single cells
followed by patch clamp recordings of whole cell TRPM4 and transient inward cationic currents.
After isomeric DSM tension recordings the data and statistics collected will be analyzed using SPSS
data analyzing software and MiniAnalysis software, synaptosoft, for different parameters.

Western blotting on DSM

tissues

Isolation of DSM Data & Statistic

single cell Analysis

DSM tissue Immunohistochemistry and

collection from immunocytochemistry
donor patient

RNA reverse transcription into

RNA extraction &
cDImmunohistochemistry and
sequencing
immunocytochemistry NA
 Novel Roles of TRP Channel in Urinary Bladder 7

Experimental method

In Vitro

The reverse transcription-polymerase chain reaction (RT-PCR) technique will be used to

differentiate the RNA from other TRP cells in tissues as Trizol reagent will be used to extract RNA
from DRG/TG neurons. These will late be copied to cDNA with a synthesis kit, most likely cDNA.
The detection of intracellular increase caused by TRP channel activation will be executed by use of
dissociated DRG neurons.

Quantifying the increase in

intracellular Ca2+ levels Radiometric
Ca2+ imaging will be used extensively.
Scanalytics software will be used to
analyze the imaging. Further Ca2+
permeability is used as an indicator of
reversal potential of TRP channels.
This is done using the replacement
method of alternating Cs+ solution
with Ca2+ based solution.

Scientific Research Work Schedule (4 years beginning Sept. 2018)

2018.9 – 2019.8 - Orientating self to Charite research model and facilities. Acquiring novel
experimental facilities and skills and enhancing my written and spoken English as well as the basic
German communication necessary for research purposes.

2019.9 – 2019.11- Completing the TRP data collection and research model and finalizing the
research timeline and experimental design with the help of research supervisor

2020.1 – 2020.6 - Data collection, experimental design optimization and commencement of

experiment

2020.7 – 2021.6 – Analyzing data from the experiment and repeated validation of data

2020.7 – 2020.12 – Summarizing experiment data, writing study report and journal articles

2021.1 – 2021.12 – Defend research findings

2022.1 – 2022.11 – Prepare for doctoral research report

Work and Learning Plan after Returning Home/China – Mainly on CSC

 Novel Roles of TRP Channel in Urinary Bladder 8

Upon the completion of this research study in Germany, I plan to return to China and get a
job in a general surgery institute or renowned research institute where I can conduct further research
and studies in in experimental Cancer Stem Cell particularly prostate cancer. In this regard my
research in transient receptor potential in the bladder will come in handy in understanding tumor
stem cell targeting agent. In addition to understanding the targeting agents and underlying processes
that cause cancer, I intend to undertake further study into the application of post-genome research
predominantly in the area of stem cells and signaling pathways.

I also intend to conduct further research into the role of specific TRP members’ role in the
development of prostate cancer and their alternative use as medical therapy agonists. In particular the
role of TRPM8 in the treatment of therapy and drug discovery is very intriguing. I believe a research
pursuit in this direction will yield significant progress towards the treatment of over-active bladder
and prostate cancer effectively. While my primary plan after returning to China is to gain
employment in an institute that allows me to conduct further cancer stem cell research and take part
in the treatment of cancer patients, if an opportunity to research TRPM8 arises, I will be glad to take
part in it.

Finally, I plan to get involved in the development of a comprehensive research methodology

and program to be implemented in my current university and perhaps in other Universities within
China. This will be a great way of implementing learned skills and best practices acquired at Charite
University. This will also go a long way in improving the Sino-Germany education and scientific
research partnership that is still in its infancy.
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References

1. Avelino, A.; Charrua, A.; Frias, B.; et al. (2012): „Transient receptor potential channels in
bladder function“. In: Acta Physiologica. 207 (1), pp. 110–122, doi: 10.1111/apha.12021.
2. Andersson, Karl-Erik; Gratzke, Christian; Hedlund, Petter (2010): „The role of the transient
receptor potential (TRP) superfamily of cation-selective channels in the management of the
overactive bladder“. In: BJU International. 106 (8), pp. 1114–1127, doi: 10.1111/j.1464-
410x.2010.09650.x.
3. Chuanzhang, Li (2013): „Research progress of the relationship between TRP ion channel family
and bladder function“. In: Shandong Medical Journal. 30 , pp. 92–95, doi: 10.3969/j.issn.1002-
266X.2013.30.041.
4. Hamilton, Kirk L. (2016): „New life in overactive bladder. Focus on ⠀œNovel regulatory
mechanism in human urinary bladder: central role of transient receptor potential melastatin 4
channels in detrusor smooth muscle function⠀“. In: American Journal of Physiology-Cell
Physiology. 310 (7), doi: 10.1152/ajpcell.00039.2016.
5. Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; et al. (2016): „Novel regulatory
mechanism in human urinary bladder: central role of transient receptor potential melastatin 4
channels in detrusor smooth muscle function“. In: American Journal of Physiology-Cell
Physiology. 310 (7), doi: 10.1152/ajpcell.00270.2015.
6. Nilius, Bernd; Owsianik, Grzegorz (2011): „The transient receptor potential family of ion
channels“. In: Genome Biology. 12 (3), p. 218, doi: 10.1186/gb-2011-12-3-218.
7. Kaneko, Yosuke; Szallasi, Arpad (2014): „Transient receptor potential (TRP) channels: a clinical
perspective“. In: British Journal of Pharmacology. 171 (10), pp. 2474–2507, doi:
10.1111/bph.12414.
8. Parajuli, Shankar P; Hristov, Kiril L; Sullivan, Michelle N; et al. (2013): „Control of urinary
bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol“.
In:Channels. 7 (6), pp. 537–540, doi: 10.4161/chan.26289.
9. Premkumar, Louis S. (2014): „Transient Receptor Potential Channels as Targets for
Phytochemicals“. ACS Chemical Neuroscience. American Chemical Society Retrieved am
11.03.2018 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240255/.
10. Wu, Guanling; Wang, Jiang; Chen, Guojung; et al. (2017): „microRNA-204 modulates
chemosensitivity and apoptosis of prostate cancer cells by targeting zinc-finger E-box-binding
homeobox 1 (ZEB1)“. In: Am J Transl Res. 9 (8).
11. Yu, Du; Ying-Jin, Shi; Yan-Hui, Zhang; et al. (2017):
„http://www.avensonline.org/fulltextarticles/JSUR-2332-4139-S1-0001.html“. In: Journal of
Clinical Medicine. 4 (77), pp. 04–77, doi: 10.13188/2332-4139.s100001.