Contractile responses of urinary bladder in an experimental model of chronic renal failure

Chronic kidney disease is a public health problem with increasing prevalence caused by diabetes, hypertension and glomerulonephritis. Number of publications investigate the lower urinary tract dysfunction due to CKD is limited. Th ere is a high incidence of bladder dysfunction of diff erent degrees in patients with renal failure. Mechanism of the lower urinary tract dysfunction in these patients is not well known. In this study, we aimed to investigate the eff ects of CKD on detrusor function in a rat model of CKD. In our study,  Wistar Albino rats have been divided into two groups as CKD and control groups. To the experiment group, left partial nephrectomy and right nephrectomy have been applied. CKD confi rmation has done with the BUN and creatinin values from the blood of the rats. Th e bladder strips were prepared from the CKD and control groups and its contractile responses were evaluated in-vitro. Th ere wasn't a considerable diff erence with the contractile responses caused by carbachol, KCL. Th ere was a considerable increase in the contractile responses caused by ATP, ADP and electrical fi eld stimulation on the behalf of the CKD group. Th e present study demonstrated that isolated DSM of CKD group showed signifi cantly increased contraction responses to purinergic agonists ADP, ATP and atropine resistant component in electrical fi eld stimulation-induced contractions as compared to those of the control group. Bladder overactivity and reduced bladder volume in CKD patients might be due to the change in purinergic system. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
The term lower urinary tract symptoms (LUTS) is now universally recognized as the preferred terminology to describe a constellation of symptoms that may be caused by multiple pathologic conditions.According to current () International Continence Society (ICS) definitions, LUTS can be divided into storage (increased daytime frequency, nocturia of at least one episode/night, urgency and UI),voiding (slow or intermittent stream during micturition, splitting or spraying of the urine stream, straining, hesitation, terminal dribble) and postmicturition symptoms (feeling of incomplete emptying and postmicturition dribble) [].With the presence of one or more of the above symptoms, the diagnosis is lower urinary tract dysfunction.A great number of topics can be mentioned in etiology of lower urinary tract dysfunction such as bladder outlet obstruction, overactive bladder syn-drome, pelvic organ prolapse, urinary tract infection, neurologic conditions, oncologic diseases, diabetes mellitus, metabolic syndrom and chronic kidney disease (CKD).Chronic kidney disease is a public health problem with increasing prevalence caused by diabetes, hypertension and glomerulonephritis [].Causal connection between lower urinary tract dysfunction with CKD is also well-known especially in pediatric population but number of publications investigate the lower urinary tract dysfunction due to CKD is limited.Th ere is a high incidence of bladder dysfunction of diff erent degrees in patients with renal failure [-].Mechanism of the lower urinary tract dysfunction in these patients is not well known.In this study, we aimed to investigate the eff ects of CKD on detrusor function in rat model of CKD.

Animals
Twenty Wistar Albino male rats weighing -g provided by Experimental Animal and Research Laboratory were used.All the experimental procedures were approved by animal experimental study local ethics committee of our institution, ethical approval number is  and all experiments were conducted in accordance with NIH guide- lines for the care and use of laboratory animals.Rats were housed at constant room temperature with -hr light and dark cycles.Food and water were available ad libitum.

Creating CKD model
Rats were divided into two groups as control (group ) and CKD group (group).Rats were anesthetized with ketamine and xylazine.Laparatomy incision and left total nephrectomy and right / partial nephrectomy were performed on CKD group.Control age and weight-matched rats (SHAM group) were submitted to similar procedures with the exception of the nephrectomy.Twelve weeks after surgery, the blood was collected for the measurement of blood urea nitrogen (BUN), creatinine and verificaton of CKD via rats' tail vein (Beckman Coulter,Synchron LX®, USA).

In Vitro Functional Studies
All rats were anesthetized with isoflurane and euthanized.Urinary bladders were removed and sectioned horizontally at the level of the ureters.Two bladder strips were prepared  x mm, taking special care to preserve the integrity of the urothelial layer during preparation.Isolated detrusor smooth muscle (DSM) strips were mounted in -ml organ baths containing Krebs-HCO solution with the following composition.(mM):  NaCl, . KCl, . CaCl, . MgSO, . KHPO,  NaHCO, and  glucose, pH ., at °C and bubbled with a gas mixture of  O and  CO.Th e bladder strips were equilibrated for approximately  min during which the buff er solution was refreshed every  min.Pretension of  gr was applied to all strips, isometric contractions were measured with a force transducer (Grass -FT  Force Displacement İtaly), and normalized based on strip cross-sectional area.Following the equilibration, the tissues were challenged with  mM potassiumchloride (KCl) for  min and contracted all strips; washed again with fresh buff er.After more bladder strips left to settle for the implementation of the agonist and antagonist substances, neurally evoked contractions were induced using electrical field stimulation (EFS) via platinum wire electrodes.

Contractile studies
Isolated bladder strips which obtained from control and CKD groups were treated with  mM KCl and responses received by KCL graphed; checked the contractility of strips before and after performing the contractile studies.Bladder strips were contracted with the cumulative concentrations of carbachol (x  - to  - M), with adenosine triphosphate (ATP) ( x  - to  - M), adenosinediphosphate (ADP) (x  - tox  - M).Bladder strips was evoked with EFS V,  msn; , , , , ,  Hz frequencies during  sec after the equilibration and responses were recorded.Before applying the EFS atropine  - M was added the organ bath.

Statistical Analysis
Data are expressed as mean ± standart deviation of  rats.Th e diff erence between groups was assessed by Student's ttest with using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, version ).Th e signifi cance of diff erences between groups was investigated by Scheff e F test.For all analyses, a p < . was considered statistically signifi cant.

RESULTS
CKD rats exhibited higher serum creatinin and BUN levels (.±.mg/dl and ± mg/dl, respectively) comparing with the control rats (.±.mg/d L and ± mg/dl) at twelfth week of operations.

DISCUSSION
LUTS are experienced by individuals with pathology aff ecting the lower urinary systemarising from detrusor overactivity, sphincteric weakness, sensory bladder disorders and prostate enlargement, and are generally divided into three groups: storage, voiding and postmicturition symptoms [].Storage symptoms (inclusive of overactive bladder, OAB) include increased urinary frequency, nocturia, urinary urgency, and urinary incontinence.Voiding symptoms include slow/ weak stream, hesitancy and terminal dripping, whereas postmicturition symptoms consist of incomplete emptying and postmicturition dripping.LUTS also encompass symptoms associated with sexual intercourse and genital and LUT pain.Prevalance of LUTS were investigated by many resarchers and many studies.In a large population-based study ( EPIC) using the  ICS definitions, Irwin et al reported that prevalence of having at least one of the LUTS, was found to be . in men and . in women aged ≥ years [].In EpiLUTS which is large population study of three countries, Coyne et al.reported that the prevalence of having at least one LUTS, at least 'sometimes' was . for men and . for women, and . and . for at least 'often' for men and women, respectively [].Th e normal micturition cycle is mostly comprised of the storage phase, with only a small minority of the time spent in actual voiding.Th e storage phase is governed by the sympathetic nerve, which causes the relaxation of the detrusor through beta- receptor mediated relaxation.Voiding in healthy situations is triggered by acetylcholine released from parasympathetic nerves activating the postjunctional muscarinic receptors in the detrusor.Urine storage reflexes are organized in the spinal cord, involving a host of neurotransmitters including norepinephrine, dopamine, serotonin, excitatory and inhibitory amino acids, ATP, nitric oxide and neuro-  peptides.Th is complex system is aff ected by many factors that hormonal, neuronal, metabolic, noxious chemichals etc. High incidence of bladder dysfunction at diff erent degrees was observed in patients with CKD.Major problems are bladder hypersensitivity, poor bladder compliance, detrusor instability and detrusor-sphincter dyssynergia in this group of patients [-].This situation is usually dependent to defunctionalized bladder and lower urinary tract dysfunction that may continue in this patients with functionalized bladder after renal transplatation (RTx) [].However, many other factors may aff ect the detrusor function on CKD patients as reduced volume of urine, vascular supply or lower urinary tract smooth muscle, irritation of urothelium.To investigate the impact of CKD on bladder function is possible in relevant preclinical models in which these other infl uences can be eliminated.Th e use of rats as animal models to study normal bladder function and experimentally induced bladder dysfunction is well established [].Van der Weide et al. [] researched  renal transplant patients using a written questionnaire,  patients (.) had urinary urgency occasionally to always.Th ey reported the incident rate of this again  years later ( years after their RTx), and . were at the same level after immediate RTx.Regarding frequent urination during the daytime and night, RTx patients also kept a high incidence  years after their operation [].Zerman et al. prospectively evaluated  patients ( women and  men, mean age of . years, ranged between - years) who suff er from CKD with non urologic reasons for lower urinary tract dysfunction before entering a renal transplantation waiting list.They reported that only  patients have normal lower urinary tract function [].Tsunoyama et al. [] researched  end stage renal disease patients ( men and  women; mean age . years; mean period of renal replacement therapy (RRT) . months).They evaluate these patients with urodynamic study before RTx.Th ey reported that  patients (.) who had detrusor overactivity before their operation and this result is quite high compared to normal Japanese population Neurogenic contractions of the bladder refl ect partly post ganglionic release of ACh and ATP, nonadrenergic, noncholinergic neurotransmitter, which are excitatory transmitters released from parasympathetic fibers [-].Detrusor smooth muscle is endowed principally with M and M muscarinic receptors with the former predominating in number.M muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile eff ects of acetylcholine in the detrusor.Emerging evidence suggests that M muscarinic receptors, via inhibition of adenyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation [].Carbachol creates contractile responses via muscarinic cholinergic receptors in vitro studies.Th e contractile responses elicited by carbachol were dose dependent and no significant difference was observed between two groups in our study.In contrast to previous studies, this fi nding of our study suggests that muscarinic system does not seem to be responsible in explanation of overactivity and decrease bladder capacity that observed in patients with CKD.Other transmitters besides acetylcholine and noradrenaline suggested to contribute to the regulation of the urinary bladder function are adenosinetriphosphate (ATP), prostaglandins and vasoactive intestinal polypeptide [].ATP generates a transient contraction caused by the stimulation of the PX purinoceptors causing depolarization through non-selective cation channels [].However in rats a signifi cant part of bladder contraction is mediated by adenosine triphosphate [].On the otherhand the sensation of bladder fullness is the primary step in the initiation of the micturition refl ex.Aff erent innervation of the bladder, producing sensations of fullness leading to detrusor contraction, is conveyed by the pelvic and hypogastric nerves, which contain myelinated (A) and unmyelinated (C) axons.Th e basis of transduction between bladder filling (mucosal stretch) and aff erent activation is chemically based mediation.Th e urothelium releases ATP at the basolateral surface when the hydraulic gradient across the bladder wall is altered by mucosal stretch and ultimately mediates aff erent excitation [].Several receptors are believed to mediate aff erent nerve fi ber excitation by generating depolarizing responses.Immunoreactivity to purinergic (PX) receptors has been located adjacent to nerve fi bers in the mouse and human bladder, in the suburothelial space and urothelium in particular [-].Our studies in isolated detrusor smooth muscle of CKD group showed greater contraction responses to purinergic agonists ADP, ATP.ATP is one such transmitter contributing to the atropine-resistant contraction by acting on the PX receptor to EFS.Contractile responses to EFS of CKD group, under cholinergic antagonist atropine, were found to be signifi cantly increased compared to the control group.In our study, signifi cantly increased contraction responses to ATP and ADP in CKD group might depend on increased number of purinergic receptors at bladder tissue.Increased response to EFS in presence atropine in CKD group also supports this assumption.It can be claimed that purinergic receptors might be upregulated due to decreased ATP release from the urothelium because decreased urine volume and reduction of the storage-discharge cycle per day, and stretch stimulation of urothelium in CKD group.Purinergic receptor density in bladder tissue from CKD group should have been investigated with immunostaining methods and the lack of these data can be considered as a limitation of this study.

FIGURE 1 .
FIGURE 1. Concentration-response curves of detrusor muscle strips to 10 -8 -10 -4 M carbachol in the control and CKD group

FIGURE 3 .
FIGURE 3. Concentration-response curves of detrusor muscle strips to 10 -6 -10 -3 M ADP in the control and CKD group