The Study Of HCN Channel’s Effects And Mechanisms In OAB Occurrence And Development

Overactive bladder (OAB) is a common bladder dysfunction. Itencompasses urinary symptoms such as frequency, urgency, and urgeincontinence. OAB has seriously affected the quality of life, including physicaland mental health of the patients, and increase the burden of social and publichealth care system. Normal bladder is in a "quiet" state during urine storage, andmaintains a low intraluminal pressure in early filling phase due to goodcompliance. All of these are conducive to the delivery of urine from the upperurinary tract and avoid urinary fluid vesicoureteral reflux. This process iscompleted via the cooperation of complex neural, humoral, mental awareness andother factors. Once the cooperation failed, lots of clinical symptoms appear, suchas the bladder spontaneous contraction, which is out of conscious control. Allthese clinical manifestations are associated with overactive bladder (OAB).Bladder pontaneous contraction is called detrusor overactivity (DO), which canbe recorded and diagnosed by clinical urodynamic study. OAB has a highincidence in the western countries, and is recognized as one of the10mostcommon chronic diseases. Although there is no exact incidence of large-scale clinical epidemiology report in China, OAB is a very common disease accordingto the situation of clinical diagnosis and treatment in daily work, So far, there isno ideal treatment for OAB. The commonly used treatment is anticholinergicdrugs. But its severe side effects limit its efficacy and patient compliance is verypoor. Therefore, it is urgent to find new and effective alternative therapies inOAB.Although there are many literatures on the pathogenesis of OAB, its exactpathogenesis has not been clearly understood. On the whole, the etiologicalfactors of OAB are complicated, and different causes lead to differentpathogenesis. The2011version of “bladder overactivity diagnostic treatmentguidelines” proposed four possible mechanisms:①detrusor instability;②bladder hyperesthesia;③urethra and pelvic floor muscle dysfunction;④otherreasons. Among them, the most common reason is detrusor instability which isoften induced by BOO disease, including BPH, urethral stricture. Although OABis a multifactorial disease, there are already considerable evidences that thestructural and functional changes of ICCs and SMCs, secondary to bladder outletobstruction, which plays a very important role in the development of OAB.It has been found that the spontaneous electrical activity of detrusororiginated from the ICCs located in the border of muscle bundles. Once thespontaneous excitation has been initiated from ICCs, it will excite bladderdetrusor to generate spontaneous action potentials and propagated to the wholebladder through gap junctions. In recent years, more attention is paid to thefunction of ICCs in physiological and pathological condition. In pathologicalcondition, it is often accompanied with the abnormality of number, distribution orfunction of ICCs, which will cause urination disorders. Such as in OAB patientsand animal models, the number of ICCs is significantly higher than normalcondition. It is reported that inhibiting the activity of interstitial cells can reducethe spontaneous electrical activity of detrusor in OAB patient and improvebladder capacity, compliance in animal experiments.Although the study of the excitability of bladder ICC is still in its infancy, HCN channels have been found in ICC of gastrointestinal tract. HCN channelsare studied more clearly than other pacemaking channels in present study and areregarded as the structural basis of the Ih current. The current of Ih, ahyperpolarization-activated current, plays a very important role in pacemaking. Itcan regulate the resting membrane potentials of some excitable cells and is thekey factor of sustaining series electrophysiological characteristics. Therefore, wesuppose that the involuntary contraction in OAB may relate to the changes ofHCN channels. These changes lead to abnormal discharge and contraction infilling phase. The reason why drugs can reduce the spontaneous electrical activityin OAB is that it can reduce the expression of HCN channels in ICCs. Thus,research that focuses on the HCN channels could be useful to study the effects ofICCs and mechanisms in occurrence and development of OAB.The main content of the thesis includes3aspects as below:Part I: Distribution and expression of HCN channels in human and ratbladderNormal bladder tissue of human and rat was chosen to detect HCN1,2,3,4channels in the mucosa, submucosa, muscularis by immunofluorescence andWestern blot methods. Furthermore, we found some vimentin and HCN4positivecells which had similar morphology with c-kit positive mast cell around bladdermicrovessels.The main results:1. The results of immunofluorescence in human bladder: c-kit positive ICCs inthe submucosa and muscularis of human bladder were rare, but vimentin-positiveICCs were widely distributed in human bladder submucosa and muscularis. Thissuggestes vimentin can be used as a reliable marker for ICC of human bladder.2. The results of immunofluorescence in human bladder: ICCs were mainlydistributed in the submucosa and muscularis of the bladder. In the muscularis,ICCs were distributed not only around the sarcolemma, but also inside the musclecells.3. The results of immunofluorescence in human bladder: HCN4positive ICCs were distributed in the bladder submucosa and muscularis. HCN4positive ICCsin the submucosa may be related to information transfering of bladder. HCN4positive ICCs in the muscularis may be related to spontaneous contractions ofbladder.4. The results of immunofluorescence in human bladder: there were some HCN4and vimentin double-labeled mast cells located in submucosa, which may berelated to the release of cytokines and active substances.5. The results of immunofluorescence in rat bladder: c-kit positive ICCs werewidely distributed in the submucosa and muscularis. This suggestes c-kit can beused as a reliable marker for ICCs. Different staining results of vimentin and c-kitin human and rat may be related with the species difference.6. The results of immunofluorescence in rat bladder: HCN4positive ICCs weredistributed in the bladder submucosa and muscularis.7. The results of IPP software analysis: the expression of HCN4channel weresignificantly higher than other subtypes either in human or rat submucosa.8. The results of Western blot analysis: the expression of the HCN4channel wassignificantly higher than other subtypes either in human or rat muscles.Conclusions:1. HCN1,2,3,4channels are all expressed in ICCs of human and rat bladder.2. The expression of HCN4channel in ICCs is higher than other subtypes inhuman and rat bladder.3. There are some HCN4and vimentin double-labeled mast cells located inhuman bladder submucosa.Part II: Distribution and expression of HCN4channel in OAB bladderThis part was designed to explore the distribution and expression of HCN4channel in OAB bladder. Male SD rats were devided into4groups: control,4weeks,6weeks,8weeks operation group. Immunofluorescence and Western blotwere used to detect HCN4channel’s distribution and expression in thesubmucosal and muscularis in different groups. And then HCN4channel was alsodetected in the cultured ICCs by immunofluorescence. The main results:1. The results of bladder urodynamic test in rat OAB model: the frequency ofspontaneous contraction was increased obviously after partial bladder outletobstruction, and8weeks group were significantly higher than other groups.2. The results of immunofluorescence in different OAB groups: the number ofICCs was increased significantly after partial bladder outlet obstruction. Thisindicates the increased expression of ICCs is an important part of pathologicalchanges in OAB bladder.3. The results of immunofluorescence in different OAB groups: HCN4channelwas increased significantly after partial bladder outlet obstruction, and theincreased HCN4channel had a positive correlation with increased ICCs.4. The results of IPP software analysis in different OAB groups: after bladderpartial outlet obstruction, ICCs and HCN4channels in submucosa were allincreased, and8weeks were significantly higher than other groups.5. The results of Western blot analysis: after bladder partial outlet obstruction, theICCs growed with the increase of the HCN4channel, and8weeks group wassignificantly higher than the other group. This suggests increased HCN4channelhas a positive correlation with increased spontaneous contraction frequency ofOAB.6. The results of IPP software analysis in cultured ICCs: after bladder partialoutlet obstruction, ICCs and HCN4channels in submucosa were all increased,and8weeks was significantly higher than other groups.Conclusions:1. ICCs and HCN4channels are increased significantly in the OAB model.2. Increased HCN4channel has a positive correlation with increased frequency ofspontaneous contraction of OAB.Part III: The mechanism of HCN4channel in the OAB pathogenesisCell culture was performed using the tissue obtained from OAB rat bladder ofdifferent operation group (4weeks,8weeks) and the normal rat bladder wastreated as the control group. Whole-cell patch clamp recordings were made with cultured ICCs in each group to detect the change of Ih.The main results:1. The results of patch clamp recordings in ICCs and detrusor: No Ih current wasrecorded in detrusor, but Ih current could be recorded in ICCs. This suggests thatICCs most likely are the pacemaker cell in bladder.2. The results of patch clamp recordings in different groups of rat OAB model:the density of Ih current was increased obviously, and8weeks group wassignificantly higher than the other group. This suggests increased HCN4channelhas a positive correlation with increased Ih current density.3. The results of patch clamp recordings in different groups of rat OAB model: inOAB group, the density of Ih current is significantly higher than normal group.This indicates that increased current density could enhance the spontaneousexcitatory current of ICC.Conclusions:1. Ih current can be recorded in ICCs. This suggests that ICCs most likely are thepacemaker cell in bladder.2. The density of Ih current in OAB group is significantly higher than normalgroup, which indicates that increased current density could enhance thespontaneous excitatory current of ICC.Main conclusions:1. HCN1,2,3,4channels are all expressed in ICCs of human and rat bladder.2. The expression of HCN4channel in ICCs is higher than other subtypes inhuman and rat bladder.3. ICCs and HCN4channel are increased significantly in the OAB models.4. Increased HCN4channel may be one of the reasons that cause spontaneouscontraction in the OAB.5. Increased current density could enhance the spontaneous excitatory current ofICC and this might be one of causes of increased spontaneous contractionfrequency in the OAB.