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.