The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor

OBJECTIVE: To describe the effect of a specific c-kit receptor inhibitor (imatinib mesylate) on human detrusor strips in vitro and guinea-pig cystometry in vivo, and to show histological data suggesting differences in the distribution of interstitial cells of Cajal (ICC)-like cells in 'normal' and overactive human detrusor, as these cells have been identified as possible mediators of spontaneous activity and excitability in bladder smooth muscle. MATERIALS AND METHODS: Specimens of human detrusor were stained immunohistochemically with a c-kit antibody. Human detrusor strips were mounted in a superfused organ-bath apparatus, and smooth muscle contraction was evoked with carbachol and electrical field stimulation in the presence and absence of imatinib mesylate. Also, guinea-pig urodynamic studies were conducted before and after i.v. administration of imatinib mesylate, and changes in bladder variables and spontaneous activity were recorded. RESULTS: Imatinib mesylate (10(-6)M) inhibited evoked smooth muscle contraction and spontaneous activity in overactive human detrusor, with less effect on normal human tissue. Imatinib mesylate (10(-5)M) improved bladder capacity, compliance, voided volumes, urinary frequency, and reduced contraction thresholds and spontaneous activity during guinea-pig cystometry. c-kit labelling showed significantly more ICC-like cells in overactive human detrusor than in normal specimens. CONCLUSION: c-kit receptor blockers have inhibitory effects on guinea-pig and overactive human detrusor, possibly via c-kit receptors on bladder ICC-like cells. This and the possibility that there are more ICC-like cells in overactive bladder suggest that the c-kit receptor may provide a novel target for treating detrusor overactivity.     

大鼠膀胱ICC样细胞与逼尿肌神经调控关系的形态学研究

目的 从形态学上探讨膀胱ICC样细胞在逼尿肌神经调控中的作用.方法 采用透射电镜观察大鼠膀胱内ICC样细胞、神经和逼尿肌细胞之间的超微结构关系.通过c-kit免疫荧光染色对大鼠膀胱ICC样细胞进行鉴定,并通过c-kit与PGP9.5免疫荧光双标观察ICC样细胞与神经的结构关系.结果 透射电镜显示,ICC样细胞与逼尿肌细胞紧密相邻处可见典型缝隙连接.在局部区域可见ICC样细胞的突起与神经末梢联系紧密.c-kit免疫荧光染色显示,大鼠膀胱内ICC样细胞主要位于黏膜下层、肌束边缘以及肌细胞间.c-kit与PGP9.5免疫荧光双标显示ICC样细胞与神经末梢在结构上关系紧密.结论 从形态学上看,膀胱内ICC样细胞具备参与逼尿肌神经调控的结构基础,进一步从功能学上予以证实将有助于全面阐明逼尿肌神经调控理论.     

The use of bowel interposed between proximal and distal ureter in urinary tract reconstruction

The replacement of lost ureter with bowel interposition is a feasible alternative in the reconstruction of the urinary tract. When adequate ureteral length is not present, interposition of a portion of small bowel between the proximal and distal ureteral segments is an option that minimizes the amount of bowel in the urinary tract. This, in turn, reduces mucus production and electrolyte absorption in the reconstructed urinary tract, and allows for more bowel to remain as part of the functioning gastrointestinal tract. This procedure allows use of the antirefluxing function of the normal ureterovesical junction when present. We have used successfully interposition of the small bowel between the proximal and distal ureteral segments for undiversion in 5 children. Generally, we strive to join urothelium to urothelium without the use of bowel in urinary tract reconstruction but when this is not feasible, bowel interposition has been a useful option.     

Immunohistochemical evidence of vanilloid receptor 1 in normal human urinary bladder

PURPOSE: Experimental and clinical evidences have shown the importance of the vanilloid receptor 1 (TRPV1) in the lower urinary tract. In humans, this receptor has been detected in nerve endings of primary sensory neurons, smooth muscle and connective tissue cells and in the rat also in the urothelium. The aim of this study is to identify, by immunohistochemistry, the cell types expressing TRPV1 in the human urinary bladder. MATERIAL AND METHODS: Specimens, obtained from normal urinary bladder by multiple biopsy and from ureter at the time of radical nefrectomy for renal cell carcinoma, were fixed and frozen. Full-thickness sections were processed for light and fluorescence microscopes. To label the TRPV1, three polyclonal antibodies were used: the anti-capsaicin receptor, the anti-VR1 (N-15) and the anti-VR1 (C-15). RESULTS: Urothelium, smooth muscle cells, mast cells and endothelium were labelled and the labelling was intracytoplasmatic. In the urothelial cells, the labelling was slightly granular. In the bladder urothelium, the superficial cells were more intensely stained than the basal and club-shaped cells. VR1-positive nerve fibers were seen running single and/or in groups in the sub-urothelium and as single varicose fibers in the muscle coat, and VR1-positive nerve endings in the urothelium. CONCLUSION: The present findings provide the evidence of the presence of TRPV1 on normal human urothelium where it could have important implications in the mechanism of action of intravesical vanilloids (capsaicin and resiniferatoxin).     

First prize: ureteral segmental replacement revisited

BACKGROUND AND PURPOSE: Long strictures of the proximal ureter are difficult to manage, and circumferential replacement with various natural and synthetic materials has been unsuccessful. We sought to use cultured autologous cells seeded onto graft material for proximal-ureteral replacement. Additionally, we wished to determine if urothelial cell-seeded de-epithelialized small bowel would generate adequate ureteral replacement. MATERIALS AND METHODS: Three sets of experiments were performed. First, autologous pig-bladder smooth-muscle and urothelial cells were expanded in culture on large sheets of multilayer small-intestinal submucosa (SIS). These sheets were then tubularized and used to replace a 5-cm segment of proximal ureter in pigs. Second, autologous cells harvested from the bladders of Beagle dogs were cultured and seeded on porcine ureteral acellular matrix, which was used to replace a 3-cm segment of ureter in dogs. Segments were wrapped in omentum to enhance vascularity. Third, a de-epithelialized small-bowel segment seeded with autologous bladder-epithelial cells was transversally retubularized (Monti) into a 4-cm ureteral replacement. Follow-up studies consisted of retrograde pyelography, serum chemistry assays, hematoxylin/eosin studies, and immunohistopathologic examination using antibodies against alpha-smooth-muscle actin and pancytokeratin AE1-AE3. RESULTS: Coculture of urinary-tract cells on large segments of SIS failed to create adequate ureteral replacement. All grafts were contracted and stenotic, with complete obstruction of the ipsilateral renal unit. Similar results were seen in the Beagles. Despite clinical obstruction and gross contraction of the graft, a circumferential muscular ureteral wall lined with multilayer transitional epithelium was present. Urotheliumseeded de-epithelialized Monti bowel segments resulted in patent ureteral replacement without hydroureteronephrosis and with normal renal function, serum electrolytes, and acid-base balance. However, bowel mucosa fully regenerated, with multilayer transitional epithelium growing adluminally in continuity with the proximal and distal anastomotic sites. CONCLUSIONS: Seeding of ureteral grafts with autologous bladder cells does not promote success in two largeanimal models using different xenogenic acellular matrices. However, muscle and urothelium regeneration occurs with ureteral acellular matrix in the dog. Urothelium-seeded de-epithelialized Monti bowel segments may be an acceptable substitute for long proximal ureteral segments. Further technical refinements are required to replace the bowel mucosa completely with normal urothelium.     

c-kit and ureteral peristalsis

PURPOSE: c-kit encodes a tyrosine kinase receptor that is required for the differentiation of a wide variety of cells during embryogenesis, including pacemaker cells of the gut. Functional expression of this tyrosine kinase receptor is required for gut peristalsis and c-kit expression has recently been documented in the adult murine urinary tract. In this study we analyzed the temporal onset of c-kit expression during ureter morphogenesis in vivo and determined if c-kit activity is essential for ureteral peristalsis in vitro. MATERIALS AND METHODS: The kidneys and ureters of gestational days 12.5 to 17.5 WT mice were isolated and frozen sections were prepared for analysis of c-kit, alpha-smooth muscle actin and uroplakin expression by immunocytochemical techniques. In addition, ureters were isolated from gestational day 15.5 mouse urogenital systems and cultured at the air/medium interface on 0.4 um pore polycarbonate membrane filters with Dulbecco's modified Eagle's medium/fetal calf serum in the presence or absence of antibodies that inhibit c-kit function. RESULTS: By gestational day 15.5 c-kit expression could be detected in a subset of renal epithelia and cells of the ureteropelvic adventitia. Prominent staining for c-kit was seen in the muscularis propria of the proximal ureter. In vitro studies demonstrated that isolated embryonic ureters acquire the ability to undergo unidirectional contractions after 3 days of culture, which is coincident with up-regulation of c-kit expression. Furthermore, incubation of isolated ureters with antibodies that neutralize c-kit activity markedly altered ureter morphology and peristalsis. CONCLUSIONS: We identified the initial expression and location of c-kit in the embryonic murine upper urinary tract. c-kit expression is up-regulated in the developing ureter prior to the ability of this tissue to undergo unidirectional contractions and c-kit function is required for the peristalsis in vitro.     

Distribution of neuropeptides, histamine content, and inflammatory cells in the ureter

OBJECTIVES: To determine the anatomic distribution of select neuropeptides (neurokinin A [NKA], substance P [SP], and bradykinin [BK]), of inflammatory cells (leukocytes and mast cells), and the histamine content in the normal swine ureter and compare the findings with regions of increased ureteral contractility. METHODS: Ureters from 10 pigs were obtained and cut into eight segments, proximally to distally. A portion of each ureteral segment was suspended in Krebs buffer (37 degrees C) and attached to force displacement transducers, and spontaneous contractility was measured for 30 minutes. A second portion was assayed for histamine, NKA, SP, and BK using enzyme-linked immunosorbent assay. A third portion was fixed in 10% buffered formalin, stained with hematoxylin-eosin, and evaluated histologically. RESULTS: Ureteral contractility was found to be highest in the most proximal and most distal regions of the ureter. Similarly, SP content was three times greater in the proximal ureter and two times greater in the distal ureter than in the midureter (P <0.05, n = 10). The total NKA and BK content were also higher in the proximal and distal ureter than in the midureter. Conversely, the histamine content was consistent throughout the ureter. Moreover, no significant difference in the distribution of inflammatory cells was identified throughout the ureter. CONCLUSIONS: The anatomic distribution of NKA, SP, and BK in the ureter corresponded to regions of increased spontaneous ureteral contractility, more specifically the proximal and distal ureter. Neuropeptides may play a significant role in ureteral contractility and may be a target for pharmacologic mediation during obstruction and stone passage.     

Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to pelvic pressure increase after obstruction

Prostanoids exert physiological effects on ureteral contractility that may lead to pressure changes and pain during obstruction. In the present study, we examined whether (1) obstruction changes the expression of the two cyclooxygenase (COX) isoforms, COX-1 and COX-2 in human and rat ureters and (2) administration of a selective COX-2 inhibitor influences the pelvic pressure change after experimental ureteral obstruction. Rats were subjected to bilateral ureter obstruction. Ureters were removed and dissected into a proximal dilated and distal non-dilated segment. RNA and protein were extracted and analyzed for cyclooxygenase expression by quantitative polymerase chain reaction and Western blotting. Human ureter samples were obtained from patients undergoing radical nephrectomy. Rat and human ureteral samples were processed for immunohistochemistry. COX-1, but not COX-2 mRNA, was readily detected in the normal rat ureter. COX-2 mRNA and protein expression was increased in the proximal dilated ureter compared to distal non-dilated ureter. This increased COX-2 expression was associated with increased urinary prostaglandin E2 (PGE2) excretion after release of obstruction. Immunohistochemistry showed increased COX-2 labeling in surface epithelium and smooth muscle layers in both rat and human obstructed ureters compared to control ureters. Furthermore, contractile PGE2-EP1 and thromboxane TP receptors were expressed in ureteral smooth muscle. Systemic treatment with the COX-2 selective inhibitor parecoxib (5 mg/kg/day) attenuated the pelvic pressure increase during obstruction. In summary, COX-2 expression is significantly increased in the ureteral wall in response to obstruction in the rat and human ureter and COX-2 activity contributes to increased pelvic pressure after obstruction.     

Clinicopathological study of intestinal smooth muscles,interstitial cells of Cajal,and enteric neurons in neonatal jejuno-ileal atresia with special reference to muscle morphometry

PurposeTo assess the thickness of the intestinal smooth muscle layer and analyze the distribution and density of interstitial cells of Cajal (ICC) and enteric neurons in the proximal and distal segments of neonatal jejuno-ileal atresia.MethodsThis is an observational study done over a period of one year in which fifteen cases of jejuno-ileal atresia were included. All the cases underwent laparotomy and resection of the atretic segment with variable portions of the dilated proximal segment and distal segment. Histopathological analysis was done on the sections taken from proximal segments (at 3 cm, 5 cm & 8 cm) and the distal segment (at 2 cm) from the atretic portion. The mean thickness of the inner circular muscle layer (ICML) and outer longitudinal muscle layer (OLML) was assessed in the above segments using image morphometry. In addition, we also analyzed the distribution and density of the ICCs and enteric neurons in the different segments using immunohistochemistry for c-kit and S-100, respectively. Controls included normal jejuno-ileal segments resected from postmortem cases (n = 7) and other nonrelated surgeries (n = 3). The findings were then compared with each-other and with normal controls.ResultsMean thickness of ICML and OLML of the proximal segments at 8 cm was significantly lower than at 3 cm and 5 cm of ileal and jejunal atresias (p ? 0.5). The mean thickness of ICML and OLML of distal segments at 2 cm was similar to the controls in all the atretic cases (p ? 0.5). The mean ICML thickness at proximal 8 cm segment was similar to the distal segment of both ileal & jejunal atresias (p = 0.06 & 0.37 respectively). The mean thickness of the OLML of the proximal 8 cm segments was significantly more than that at the distal segment (p = 0.008) in ileal atresias but was similar in cases of jejunal atresias (p = 0.07). Both the proximal and distal segments of ileal as well as jejunal atresias showed reduction in distribution and density of ICCs, as compared to normal controls. The density of ICCs in proximal segments at 3 cm and 5 cm was similar in both ileal (p = 0.33) and jejunal segments (p = 0.41) but was significantly lower than the proximal 8 cm segments (p ? 0.05).The distribution of ICCs in the proximal segment at 8 cm was similar to the distal segments (p ? 0.05). S-100 staining showed dense expression of neurons and glial cells with presence of submucosal giant ganglia within the proximal dilated segments as compared to the distal segments and the controls, which were more marked at 3 cm and 5 cm levels than at 8 cm level.ConclusionMuscle morphometry using image analysis is a simple technique to assess the thickness of the intestinal smooth muscle layers. There is significant smooth muscle hypertrophy along with marked alteration in density and distribution of ICCs and ENS in the dilated proximal segments up to 5 cm, and relatively milder changes at 8 cm levels, as compared to the distal segments and the controls.Type of studyPrognosis study.Level of evidenceLevel II.     

Longitudinal and thickness measurement of the normal distal and intravesical ureter in human fetuses

PURPOSE: We define reference data concerning the development of the ureterovesical junction in fetuses and newborns by measuring the diameters of the distal mesenchymal and muscular ureteral walls as well as intravesical ureteral length. MATERIALS AND METHODS: A total of 90 normal fetal and newborn ureters were investigated. Our histological studies were based on "plastinated" sections of whole pelves which allow study of the sectional anatomy of the distal and intravesical ureter. The development of the mesenchymal and smooth muscle growth of the distal and intravesical ureter was examined. The ureteral measurements were correlated with age of gestation. RESULTS: The length of the intravesical ureter and mesenchymal as well as smooth muscle walls increased in a linear mode. Significant correlations (p <0.0001) were found between gestational age and the growth of the mesenchymal as well as smooth muscle walls in the distal intravesical ureter as well as the length of the intravesical ureter. CONCLUSIONS: Significant positive linear relationships exist between gestational week, and distal and intravesical ureteral wall thickness of the mesenchymal and smooth muscle growth to the length of the intravesical ureter in fetuses and newborns. The ratio of the intravesical ureteral length-to-ureteral diameter is obviously lower than assumed previously. Data from this study can be used for a more accurate assessment of cases with abnormal lower urinary tract development.     

Effects of imatinib mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the guinea-pig urinary bladder

AIMS: In the gastrointestinal tract, slow wave activity in smooth muscle is generated by the interstitial cells of Cajal (ICC). Detrusor smooth muscle strips of most species show spontaneous contractions which are triggered by action potential bursts, however, the pacemaker mechanisms for the detrusor are still unknown. Recently, ICC-like cells have been found in guinea-pig bladder, using antibodies to the c-kit receptor. We have investigated the effects of Glivec, a c-kit tyrosine kinase inhibitor, on spontaneous action potentials in guinea-pig detrusor and intravesical pressure of isolated guinea-pig bladders. METHODS: Changes in the membrane potential were measured in guinea-pig detrusor smooth muscle using conventional microelectrode techniques. Pressure changes in the bladder were recorded using whole organ bath techniques. RESULTS: Smooth muscle cells in detrusor muscle bundles exhibited spontaneous action potentials, and spontaneous pressure rises occurred in isolated bladders. Glivec (10 microM) converted action potential bursts into continuous firing with no effects on the shape of individual action potentials. Glivec (>50 microM) reduced the amplitude of spontaneous pressure rises in the whole bladder in a dose dependent manner and abolished spontaneous action potentials in detrusor smooth muscle cells. CONCLUSIONS: The results suggest that ICC-like cells may be responsible for generating bursts of action potentials and contractions in detrusor smooth muscle. Drugs inhibiting the c-kit receptor may prove useful for treating the overactive bladder.     

Distribution of P2X receptors in the urinary bladder and the ureter of the rat

PURPOSE: One means of assessing the presence of purinoceptors and their possible participation in signaling events in tissues is through use of specific antibodies and immunohistochemical methods. A thorough immunohistochemical screening for the presence of P2X receptors on bladder and ureter sections has been performed. MATERIALS AND METHODS: Distribution of P2X receptor subtypes in rat bladder and ureter has been investigated using specific polyclonal antibodies to P2X1 through to P2X7 receptor subtypes with immunohistochemical methods. RESULTS: In both the bladder detrusor muscle and the ureteral muscle (as well as the accompanying arteries) P2X1 immunoreactivity was associated with the smooth muscle membranes. Non-membrane associated smooth muscle reactivity was seen with P2X2> P2X5 = P2X6. P2X3 immunoreactivity was seen within nerve bundles in detrusor muscle only. The fine capillary network supplying bladder and ureter smooth muscle and lamina propria was visualized with P2X4 immunoreactivity, membranes of urothelial cells gave a strong reaction with P2X5, whereas P2X6 immunostained the thin basement membrane beneath the urothelium. Nuclear staining was seen with P2X7 in the urothelium but more prominent in the bladder than in the ureter. CONCLUSIONS: Having established the distribution of P2X receptors in normal animal bladder and ureter tissue, it is now possible to perform comparable investigations on normal and diseased human tissue to establish a possible role of P2X receptors in pathogenic events.     

Characterization of the spontaneous electrical and contractile activity of smooth muscle cells in the rat upper urinary tract.

PURPOSE: We morphologically and electrophysiologically identified the cells that generate the electrical activity underlying the peristaltic contractions of the rat upper urinary tract. MATERIALS AND METHODS: Electron microscopy and tension recording techniques were used to characterize the smooth muscle cells underlying spontaneous contractions in the wall of the rat ureter, and proximal and distal renal pelvis. Intracellular microelectrodes, containing 4% neurobiotin were used to record data from the cells of the renal pelvis, which were later viewed on a confocal microscope. RESULTS: Spontaneous myogenic contractions (average 22.3 +/- 2.2 minutes(-1)) originated in the proximal renal pelvis and propagated into the distal renal pelvis and ureter in 6 preparations. Smooth muscle cells in the renal pelvis and ureter were typical in appearance with greater than 85% of their sectional area containing clumped contractile filaments. In contrast, contractile fibrils occupied only 65% of the sectional area of the smooth muscle cells within the most proximal region of the renal pelvis (pelvicaliceal junction). In strips of the renal pelvis spindle shaped cells 83 to 200 microm. long fired spontaneous action potentials (6 minutes(-1)) consisting of an initial spike, a quiescent plateau phase and abrupt hyperpolarization to a peak diastolic potential of -60 mV. Other spindle shaped cells 94 to 112 microm. long displayed small membrane transients (15 minutes(-1)) 9 to 19 mV. in amplitude, firing from a diastolic potential of -40 mV. CONCLUSIONS: It is likely that the spontaneous contractile activity of the rat upper urinary tract arises from the discharge of action potentials in typical smooth muscle cells of the proximal renal pelvis that are directly driven by the spontaneous membrane oscillations of atypical smooth muscle cells.     

Interstitial cells of Cajal in the human normal urinary bladder and in the bladder of patients with megacystis-microcolon intestinal hypoperistalsis syndrome

OBJECTIVE: To investigate the distribution of c-kit-positive interstitial cells of Cajal (ICCs) in normal bladder and bladders from patients with megacystis-microcolon-intestinal peristalsis syndrome (MMIHS, a rare congenital and generally fatal cause of functional intestinal obstruction in the newborn), the most characteristic feature of which is abdominal distension caused by a distended unobstructed urinary bladder. PATIENTS AND METHODS: Full-thickness bladder specimens were obtained from four infants with MMIHS and four controls, and processed as paraffin-wax and frozen sections. Sections were assessed using single immunohistochemistry with monoclonal and polyclonal anti-c-kit antibodies. Anti-alpha-smooth muscle actin (SMA) antibody was used to investigate the contractile apparatus in smooth muscle cells of the urinary bladder. Specimens were examined using light and confocal scanning microscopy. RESULTS: There were many c-kit positive ICCs in the normal urinary bladder, appearing as small, long, bipolar cells with only two long and several short processes. In contrast, ICCs were absent in the MMIHS bladder. alpha-SMA immunoreactivity was lower in MMIHS urinary bladder than in control sections. CONCLUSION: This study shows for the first time the presence of c-kit-positive ICCs in the normal human urinary bladder. The lack of ICCs in the MMIHS bladder may contribute to the voiding dysfunction in this disease.     

Altered Expression of Interstitial Cells of Cajal in Congenital Ureteropelvic Junction Obstruction

PurposePeristaltic contractions in the upper urinary tract serve to move urine from the kidney through the ureter to the bladder. Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. To our knowledge the pathophysiology of UPJ obstruction is unknown. C-kit positive interstitial cells of Cajal (ICCs) are pacemaker cells that facilitate active propagation of electrical events and mediate neurotransmission. We investigated the expression of c-kit positive cells in the muscle layer of normal and obstructed UPJ specimens.Materials and MethodsA total of 19 human formalin fixed, paraffin embedded specimens of intrinsic UPJ obstruction from children with a mean age of 2.3 years (range 2 months to 12 years) and 7 control samples from children with a mean age of 4.5 years (range 11 months to 9 years) were investigated immunohistochemically for the expression of c-Kit oncoprotein and peripherin by light and laser scanning microscopy. Quantification of immunolabeled structures was quantified using computerized image analysis.ResultsPeripherin immunoreactivity was strong in the muscle layer of normal UPJ specimens, while in UPJ obstructed specimens there was a decrease in peripherin positive nerve fibers. In normal UPJ specimens there were many c-Kit positive ICCs between the muscle bundles. The density of ICCs was markedly decreased in the muscle layers of UPJ obstructed specimens.ConclusionsTo our knowledge this study shows for the first time the immuno-expression of c-Kit positive ICCs in the proximal part of the normal human upper ureter. The altered density of c-Kit positive cells in UPJ obstruction may have a role in the failure of transmission of peristaltic waves across the UPJ.     

Characterization of ureteral dysfunction in an experimental model of congenital bladder outlet obstruction

PURPOSE: Ureteral dysfunction is a significant sequela of congenital bladder outlet obstruction. However, the structural and functional alterations associated with ureteral dysfunction are not well defined. A model of fetal bladder obstruction in sheep was used to characterize the changes in ureteral smooth muscle, extracellular matrix (ECM) and functional properties in response to bladder outlet obstruction. MATERIALS AND METHODS: Partial bladder outlet obstruction was created in fetal sheep at gestational age 95 days via placement of a metal ring around the proximal urethra as well as ligation of the urachus. Ureters were harvested at 109 and 135 days (full term = 140 days) to determine the relative composition of smooth muscle, ECM and urothelium by morphometric analysis and to measure DNA and protein concentrations. Ureteral tissue from 135 day gestation obstructed and control sheep was harvested and immediately placed in Krebs solution. Smooth muscle strips (2-3 mm. x 7-8 mm.) were suspended in organ baths. The frequency and amplitude of spontaneous ureteral contractions was as well as the response to electric field stimulation (EFS) were determined. RESULTS: Bladder outlet obstruction caused a significant increase in ureteral weight, smooth muscle mass and total ECM at both 109 and 135 days gestation. Total ureteral DNA was greater in obstructed compared with sham ureters at 135 days gestation. Obstructed ureters demonstrated greater amplitude and frequency of spontaneous contractions as well as more pronounced response to EFS when compared to sham ureters. CONCLUSIONS: The fetal ureter responds to bladder obstruction with smooth muscle hyperplasia and hypertrophy which is associated with increased spontaneous activity and augmented response to EFS. ECM content is markedly increased indicating a shift in the balance of connective tissue synthesis and degradation. Congenital post-obstructive ureteral dysfunction therefore appears to be the result of dysregulated smooth muscle cell growth and altered ECM homeostasis producing abnormal ureteral contractility.     

Intrinsic expression of Th2 cytokines in urothelium of congenital ureteropelvic junction obstruction

BACKGROUND: The cytokine-producing ability of urothelium, a urinary tract barrier between urine and underlying connective tissue, may exacerbate the pathogenesis of congenital ureteropelvic junction obstruction (UPJ-O) disease. A role for urothelium in human urinary tract obstruction has rarely been described. In this study, we investigated the immunopathologic characteristics of, and cytokine production by, urothelium in children with congenital UPJ-O. METHODS: Twenty-four children with congenital UPJ-O who had received pyeloplasty were enrolled. Morphologic abnormalities and pathologic and inflammatory changes of UPJ-O segments were studied. Expression of cytokines and chemokines in urothelium was investigated and compared with control tissue by immunohistochemistry (IHC), in situ hybridization (ISH), and urinary enzyme-linked immunosorbent assay (ELISA). RESULTS: Atypical or simple hyperplasia of the urothelium with evidence of Ki67 over-expression was found frequently in UPJ-O. There were variable degrees of inflammatory cell and eosinophil infiltration. Augmented expression of IL-5 and eotaxin detected by IHC and ISH, and enhanced degranulation of tissue mast cells were observed in the urothelium of UPJ-O segments. IL-4, IFN-gamma, and TNF-alpha were undetectable. Significantly higher levels of urinary IL-5, IFN-gamma, and eotaxin were detected in urine collected from the obstructed kidney. Among these, high urinary IL5 and/or IFN-gamma levels were associated with more severe obstructive uropathy (P < 0.05). CONCLUSION: Urothelium, like intestinal and respiratory epithelia, plays an active role in immunoregulation and may contribute to exacerbation of the pathogenesis of congenital UPJ-O. Many eosinophil-associated disease cytokines, which can lead to the degranulation of mast cells, are predominant regulators in UPJ-O urothelium.     

Urinary tract pacemaker cells: current knowledge and insights from nonrenal pacemaker cells provide a basis for future discovery

Coordinated ureteric peristalsis propels urine from the kidney to the bladder. Cells in the renal pelvis and ureter spontaneously generate and propagate electrical activity to control this process. Recently, c-kit tyrosine kinase and hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) were identified in the upper urinary tract. Both of these proteins are required for coordinated proximal to distal contractions in the ureter. Alterations in pacemaker cell expression are present in multiple congenital kidney diseases, suggesting a functional contribution by these cells to pathologic states. In contrast to gut and heart pacemaker cells, the developmental biology of ureteric pacemaker cells, including cell lineage and signaling mechanisms, is undefined. Here, we review pacemaker cell identify and function in the urinary pelvis and ureter and the control of pacemaker function by Hedgehog-GLI signaling. Next, we highlight current knowledge of gut and heart pacemaker cells that is likely to provide insight into developmental mechanisms that could control urinary pacemaker cells.     

Co-localization of androgen receptor with estrogen receptor beta in the lower urinary tract of the male rat.

PURPOSE: Androgens and estrogens influence voiding. In this study their target sites in the lower urinary tract of the male rat were identified. MATERIALS AND METHODS: Cryosections of the bladder body, bladder neck, prostatic urethra, mid proximal urethra and prostatic autonomic ganglia of adult male rats were immunostained with specific estrogen receptor alpha, estrogen receptor beta (ERbeta) or androgen receptor (AR) antibodies. The sections were then examined under conventional, fluorescence or confocal fluorescence microscopy. RESULTS: Co-expression of AR and ERbeta in the urothelium, bladder smooth muscle cells, proximal urethra striated muscle cells and neurons in the autonomic ganglia of the prostatic plexus suggests that estrogen and androgen have direct effects in the lower urinary tract. CONCLUSIONS: The local interaction of AR and ERbeta in the hormonal control of voiding is an intriguing possibility.