Molecular And Functional Expression Of TRP Channels In Bladder Mucosa And The Role Of Urinary ATP In Diagnosis Of BOO In BPH Patients

Background:In addition to the traditional barrier function,bladder mucosal epithelium(urothelium)has been found to express various ion channels and receptors such as transient receptor potential channels(TRP channels),Pannexin 1 channel,Piezo 1 channel,and cholinergic receptors,purinergic receptors,etc.It can response to mechanical,chemical,temperature stimulation,and release neurotransmitters such as ATP,NO,acetylcholine(Ach)and a variety of cytokines and nerve growth factors.Thus,the urothelium has been considered to be a sensory organ.The urothelium normally responses to mechanical stretch produced during bladder filling,however,the exact underlying mechanisms for mechanical transduction has not been clear yet.It has been revealed that mechanical stretch during bladder filling could lead to the increase of intracellular Ca2+([Ca2+]i)in urothelial cells and the release of ATP,which in turn activate P2X3 receptors on the sensory terminals,this process has been considered an important mechanism of bladder filling sensation.However,the cellular and molecular mechanisms of mechanical stimulation induced[Ca2+]i increase in urothelium are still unclear.Among the mechano-sensitive TRP channels expressed on urothelium,TRPV4 is most abundantly expressed.Some studies believe that activation of TRPV4 channels caused by stretch stimulation can trigger a series of physiological responses,which plays an important role in the generation of bladder filling sensation and the initiation of urination reflex.Activation of TRPV4 could induce[Ca2+]i increase and ATP release in urothelial cells.However,the intracellular mechanisms leading to the increase of[Ca2+]i and ATP release after activation of TRPV4 during bladder filling is not yet clear.To elucidate the possible mechanisms,we cultured rat urothelial cells,and a rapid mechanical stimulation was applied to the urothelial cells by making an air-liquid interface.The[Ca2+]i increase evoked by mechanical stimulation was recorded and various channels and pathways that possibly involve in[Ca2+]i increase has been explored.Method:bladders of 2-3 months Sprauge-Dawley rats were used for urothelial cells culture in vitro.After 2 days of culture,the cell was given a rapid mechanical stimulation by making an air--liquid interface.[Ca2+]i increase was recorded by calcium imaging method,blockers of membrane channels or intracellular signaling molecules were used to explore their involvement in the progress.Result:(1)The mechanical stimuli were able to induce[Ca2+]i increase,and this responses were repeatable;(2)The[Ca2+]i increase could induce ATP release;(3)Immunohistochemistry showed extensive TRPV4 expression on urothelial cells;(4)[Ca2+]i increase could be blocked(74.8%)by the non-specific mechanical channel blocker GdCl3(100 ?M);(5)[Ca2+]i increase was significantly reduced(35.7%)by Ruthenium Red(10 ?M),a non-specific TRP channel blocker;(6)[Ca2+]i increase was significantly reduced(41.1%)by HC067047(10?M),a TRPV4 specific antagonist;(7)[Ca2+]i increase was significantly reduced(71%)by 2-APB(10?M),an blocker of IP3 receptors on endoplasmic reticulum(ER).Conclusion:TRPV4 are the main membrane channels responsible for the[Ca2+]i increase induced by mechanical stimulus produced at air-liquid surface on bladder urothelial cells,intracellular Ca2+release from ER by activation of IP3 receptors are the main source of[Ca2+]i increase.TRPV4 may couple with IP3 receptors on ER to involve in the mechanical responses of urothelial cells.Background: The bladder mucosa contain urothelium and suburothelium(also called lamina propria)layer.Bladder mucosa has been considered to be both a protective layer of the bladder and a sensory receptor.Previous studies on the sensory function of the bladder mucosa have been focusing on the role of urothelium and the nerve terminals in lamina propria.In recent years,studies suggested interstitial cells(ICs)in suburothelial may regulate sensory afferents function of the bladder,but the specific mechanisms are unclear.TRP channels expressed on suburothelial ICs may have roles in modulation of bladder sensory transduction.To our knowledge,most of the studies that focused on ICs in lamina propria were from animal models,and almost all of the studies on TRP channels in ICs did not examine the functional expression level,but only examined the mRNA or protein expression level of TRP channels.Object: To explore the physiological relevance of TRP channels on ICs in bladder sensory signaling by studying the functional expression of TRP channels in ICs present in upper lamina propria.Method :(1)Tissue immunofluorescence was conducted to show the expression of TRP channels in ICs of human bladder;(2)Primary cell culture technique was used to isolate and purify bladder ICs from human bladder sample,and cellular immunofluorescence technique was performed to further confirm TRP channel expression on ICs.(3)Ca2+ imaging method was used to observe the functional expression of TRP channels on human ICs.Result:(1)Tissue immunofluorescence showed that there were a large number of Vimentin-positive ICs(Vimentin is ICs-specific marker)in the suburothelial and between detrusor bundles of human bladder,and almost all ICs in upper lamina propria expressed a-SMA;(2)Cellular immunofluorescence showed that ICs from human bladder expressed Vimentin,a-SMA and TRPA1 and TRPV4,indicating that the cells were mainlyfrom upper lamina propria;(3)Calcium imaging experiments showed that AITC and cinnamaldehyde(CA),the two specific agonists of TRPA1,could evoke intracellular calcium increase and ATP release in ICs,and this activation could be partially blocked by the specific TRPA1(HC030031)antagonist.Meanwhile,GSK1016790 A,a TRPV4 specific agonist also could evoke intracellular calcium increase in ICs and ATP release,and the activation could be blocked by the specific TRPV4 antagonist(HC067047).The above results indicate that ICs in upper lamina propria express functional TRPA1 and TRPV4 channels,and activation of these channels could induce ATP release.Conclusion : Our results suggest ICs could response to both chemical and mechanical stimuli in lamina propria and may have bidirectional communication with surrounding urothelial cells and sensory nerve endings,thus,play an indispensable role in bladder sensory afferent signaling in lamina propria.Background: Benign prostatic hyperplasia(BPH)occurs commonly in older men,with historical reports of up to 50% prevalence in men over the age of 50 years.An enlarged prostate may anatomically compress the urethra,causing bladder outlet obstruction(BOO).This results in lower urinary tract symptoms(LUTS),which include voiding symptoms and storage symptoms.However,studies indicated that the international Prostate Symptom Scores(IPSS)might not be helpful in the diagnosing of patients with BOO and predicting its severity.Urodynamic study(UDS)is the gold standard for the evaluation of BOO and the degree of obstruction.However,USD is time-consuming and invasive,and it can be expensive for some patients.Additionally,some hospitals may not be able to afford the urodynamic equipment.All of these reasons require a non-invasive and less expensive test or a biomarker to evaluate the existence of BOO and the degree of obstruction in BPH patients.Traditionally,the urinary bladder urothelium was considered to be a passive membrane,but it has been reported to have sensory neuronal-like properties and it responds to mechanical and chemical stimuli by releasing transmitters or mediators including ATP,ACh,and PGE2.Released ATP will activate P2X3 receptors on the suburothelial afferents to trigger the micturition reflex.Under some pathological conditions,the urothelium releases more ATP into the lumen,and the urinary ATP concentration was found to be increased in patients with overactive bladder,interstitial cystitis,and bladder infection or inflammation.Thus,urinary ATP was suggested as a biomarker for these situations.In addition to the stretch stimulation during bladder filling,intravesical pressure during bladder isometric contraction could further stimulates ATP release from urothelium.In support of this idea,the urinary ATP concentrationin BPH patients has been found to be higher than asymptomatic controls and urinary ATP has been suggested to be a non-invasive biomarker of BOO.However,the diagnostic value of urinary ATP for BOO require validation by urodynamic tests t.For BPH patients in the compensated stage,the intravesical pressure during voiding would increase as the degree of BOO increased and more ATP may be released.Therefore,the degree of BOO might be inferred by measuring the urinary ATP concentration.Object: In this study,the degree of BOO was quantified using the BOO index(BOOI)in BPH patients who underwent urodynamic exams,and we aimed to explore the possibility that urinary ATP level could be a non-invasive biomarker for bladder outlet obstruction(BOO)as well as its severity in BPH patients by analyzing the correlation between BOOI and urinary ATP..Methods: We included 117 BPH patients who underwent urodynamic studies and 109 asymptomatic controls.Urine samples at normal desire(from patients and controls),instilled fluids at maximum cystometric capacity(capacity fluid)and voided fluids during pressure-flow study(only from patients)were collected.ATP concentration of collected samples was measured with luciferin-luciferase bioluminescence assay and normalized by urine creatinine(ATP/Cr).The degree of BOO was quantified with BOO index(BOOI).Correlation between urodynamic parameters and urinary ATP concentration was analyzed in BPH patients.Results: Urinary ATP concentration of BPH patients was significantly higher than controls(ATP:118.54±7.18 vs 47.67±2.42nM;ATP/Cr:126.20±7.99 vs 51.8±2.63pmol/mg,p<0.001).For BPH patients,a significant positive correlation was found between urinary ATP concentration and BOOI(r=0,5441,p< 0.0001).Although BPH patients with detrusor overactivity or a history of acute urinary retention had increased urinary ATP,significant positive correlation between ATP and BOOI was also observed in these patients.When BOOI greater than 40 was set as a cutoff point to differentiate BOO from NO-BOO patients,the area under the ROC curve was 0.77(95%CI:0.67-0.88,p<0.001).Conclusions: BPH patients with BOO release higher amounts of ATP intourine.Urinary ATP can be used as a non-invasive biomarker of BOO,its level may also have a predictive value for obstruction degree.