Discussion
This study shows the functional effect of exogenous CS instillations on
urothelial barrier function after repeated damage with protaminein-vitro over the course of several days, to mimic the
chronically impaired urothelium in patients suffering from BPS/IC.
Protamine instillation impaired relevant barrier markers such as tight
junction formation as was shown by immunohistochemistry.
Urothelial production of CS containing proteoglycans was confirmed with
the IF assay and instillation of exogenous CS increased this signal
clearly. The SEM analysis showed that CS instillation creates a mucous
like covering layer on the urothelium, implying adherence of CS to the
urothelium.
Although BPS/IC is a chronic condition, most known in-vitro andin-vivo studies focus on the acute effects of damage on the
urothelium.[15][16][36][37] By using a chronic
inflammatory in-vitro model, we were able to qualitatively
investigate evident features of GAG therapy being barrier function
enhancement and barrier recovery.
The culturing protocols and in vitro set-up of the experimental
demonstrated a high degree of reproducibility.[15][16] TEER
measurements can be conducted repeatedly in time without altering cell
health and barrier function. To qualify as a representative urothelial
barrier model, terminal differentiation is essential and this feature
lacking in many preclinical studies who often use undifferentiated cell
lines. TEER measurements demonstrate how much barrier properties vary
between undifferentiated (100 Ω.cm2) and
differentiated (>1000 Ω.cm2 ) urothelial
layers and shows how tight a urothelial barrier is compared to other
epithelia.[26]
Our in vitro model shows evident plasticity of urothelial cells with a
remarkable recovery rate after multiple damaging events. Nonetheless,
the TEER levels in the protamine alone treated samples remained
approximately 5-6 times lower compared to pre-treatment throughout the
experiments, corresponding to a chronic damaged barrier. This was also
supported by the immunohistochemistry results that showed a decreased
signal for adherence junctions, tight junctions and chondroitin sulfate
which is comparable to the histology as is seen in BPS/IC.[6]
Experiments show that CS instillations increase the regeneration of
urothelial barrier properties and thereby improves urothelial recovery
after damage. CS treatment resulted in complete recovery of barrier
after 3 days and even higher TEER. Results also show that CS treatment
has a relatively larger effect on barrier repair after multiple
protamine hits compared to controls. This could imply that it has a
larger therapeutic effect in bladders with chronic urothelial damage. IF
analyses showed the urothelium surface was covered with CS after
treatment. This finding is in line with the animal study by Kyker et al.
that showed adherence of CS to damaged urothelium, by installing
labelled CS in damaged mouse bladders.[15] Hauser et al. also showed
recovery of the barrier function after intravesical CS in a rat model of
bladder damage as measured by permeability to a potassium ion
mimetic.[16] Our results do not clarify what an optimal dosing
scheme would be for clinical CS treatment.[17, 18]
The experimental data does not demonstrate a direct effect of CS on
barrier (TEER). CS-treatment protects cells against the secondary
harmful effects after a damaging event, possible hypothesis could be
preventing exposure of cell membranes to hazardous ions or molecules. Or
it could stimulate barrier regeneration via an undetermined signalling
cascade. A recent publication by Rooney et all showed that HA+CS
treatment stimulates the endogenous production of chondroitin sulfate in
cultured urothelial cells. This effect on endogenous production of GAGs
has not been investigated for CS separately thus far. [19]
Inflammatory markers or growth factors were not evaluated in this study,
but Stellavato et all also showed a reduction of interleukins (6 & 8)
after GAG therapy.[20]
The in-vitro model offers the possibility to investigate the
effects of therapy on the functional barrier of damaged urothelium that
can be measured more precisely compared to in vivo models because it
uses fixed surface areas and controlled cell densities. So far, this has
only been conducted for CS, but other intravesical compounds for BPS/IC
such as heparin, hyaluronic acid and pentosan polysulfate could also be
analysed and compared to each other.[21] Although a single
randomized study showed an advantage for CS instillations compared to HA
in BPS/IC.[22] To our knowledge, CS is the only sulphated GAG
observed in the luminal layer of healthy urothelium, although presence
of heparan sulfate was detected in deeper layers of the
urothelium.[9] Especially the functional barrier properties of
hyaluronic acid and pentosan polysulfate deserves more attention since
these are also commonly used in daily clinical practice for BPS/IC
treatment.