Changes in interstitial cell of Cajal-like cells density in congenital ureteropelvic junction obstruction

Purpose  

The authors examined the number of interstitial cells of Cajal-like cells (ICC-LCs) in obstructed ureteropelvic junction (UPJ) in comparison with normal UPJ specimens and age-related changes.     

Structural changes of smooth muscle in congenital ureteropelvic junction obstruction

Background/Purpose

Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis. Previous studies have reported that the excess amount of collagen restricting mobility and resiliency of the UPJ is the result of an impaired collagen production by anomalous smooth muscle cells (SMCs). Our purpose was to evaluate the role of SMC differentiation in the pathogenesis of UPJ obstruction.

Methods

Surgical specimens of UPJ from 21 patients (8 girls/13 boys) who were subjected to dismembered pyeloplasty were examined immunohistochemically using monoclonal antibodies against smooth muscle (SM) myosin heavy chain isoforms including SM1, SM2, and SMemb. The age ranged from 1 month to 13 years. Ureteropelvic walls taken from 14 forensic autopsy cases, with no urological abnormalities, served as age-matched control group.

Results

The immunohistochemical expression of SM1 and SM2 in UPJ obstruction was significantly increased when compared with controls (P < .05). In contrast, there was no statistical difference of expression of SMemb.

Conclusion

Our findings supported the hypothesis that the primary anomaly in UPJ obstruction may be attributed to a malfunction of SMCs in the ureter.     

The distribution of interstitial cells of Cajal in congenital ureteropelvic junction obstruction

Purpose

The authors analysed the distribution of c-kit-positive interstitial cells of Cajal (ICCs) in obstructed ureteropelvic junction (UPJ) and its age-related changes.

Methods

Twenty specimens were obtained from children with intrinsic ureteropelvic junction obstruction (UPJO), at the average age of 8.1 years (8 months–16.8 years), fixed in formalin and embedded in paraffin. Five control samples were taken from children at the average age of 2.3 years (2.4 months–7.4 years). All specimens were analysed by the immunohistochemistry test with light microscopy with respect to c-kit expression. The distribution of c-kit-positive ICCs in the two groups was compared and the correlation between the distribution of c-kit-positive ICCs and the patients’ age in UPJO cases was analysed. The results were examined by Yates’ χ² test, Mann–Whitney U test, and t test for Pearson’s correlation coefficient. A P value < 0.05 was considered as statistically significant.

Results

No statistically significant differences were found in the distribution of c-kit-positive ICCs between UPJO and the control group. No correlation was established between the age of patients with UPJO and the distribution of c-kit-positive ICCs.

Conclusion

No distributional difference found in obstructed and unobstructed UPJ seems to indicate that UPJO is not associated with anomalous distribution of c-kit-positive ICCs. Age-related changes in the expression of c-kit-positive ICCs are equally distributed in obstructed UPJ.     

Thickness of the renal pelvis smooth muscle indicates the postoperative course of ureteropelvic junction obstruction treatment

ObjetiveTo investigate the relationship between the histopathologic findings and the postoperative course of children surgically treated for ureteropelvic junction (UPJ) obstruction.Material and methodsTwenty-eight patients operated for unilateral UPJ obstruction from 1998 to 2005 with adequate histopathologic specimens and postoperative follow up were retrospectively reviewed. Specimens were stained using elastic van Geisson to differentiate smooth muscle from collagen and elastin. Postoperative follow up included renal ultrasound (U/S) and diuretic renogram studies.ResultsTwelve patients with mean renal pelvis smooth muscle thickness (mRPSMT) of 136.97 ± 34.17 improved on the 6^th postoperative month. Nine patients that improved after 9 months postoperatively had mRPSMT=173.61 ± 33.91. The rest 7 patients that improved on the 12^th postoperative month had mRPSMT=258.78 ± 96.09. Correlation between renal pelvis smooth muscle and time of postoperative improvement was extremely significant (r = 0.7928, p < 0.0001).ConclusionThe thickness of the renal pelvis smooth muscle is significantly correlated to the postoperative course of patients with UPJ obstruction and can be used as a prognostic tool for the onset of their improvement.     

Ion-channel currents of smooth muscle cells isolated from the prostate of guinea-pig

OBJECTIVE: To characterize the voltage-activated ion-channel currents in guinea-pig prostate smooth muscle cells (GPSMCs). MATERIALS AND METHODS: GPSMCs were isolated using collagenase, and used in a whole-cell patch clamp study. RESULTS: When GPSMCs were dialysed with a CsCl solution all the outward K+ currents were blocked and the step-like depolarization (holding voltage -70 mV) of the cell membrane evoked inward currents that were completely blocked by nifedipine (1 micromol/L). With KCl solution, step depolarizations showed outward K+ currents composed of fast, transient outward current (Ito) and outward currents that did not inactivate. Ito was resistant to a high concentration of tetraethylammonium (TEA, 5 mmol/L) but was blocked by 4-aminopyridine (5 mmol/L). The half-activation and half-inactivation voltages of Ito were 6 mV and -58 mV, respectively. With low Ca2+ buffer (0.1 mmol/L EGTA) in the solution, there were spontaneous transient outward currents (STOCs) at depolarized membrane voltages (0 mV). STOCs were blocked by TEA (1 mmol/L) or iberiotoxin (10 nmol/L) but were insensitive to apamin (100 nmol/L). CONCLUSION: This voltage-clamp study showed that GPSMCs have l-type Ca2+ channels and more than two types of K+ channels. The voltage- and time-dependent changes of these ion channels and their interactions might be important in forming action potentials and regulating contractility.     

Studies on isolated smooth muscle cells. IX. Application of papain for isolation of single smooth muscle cells from guinea-pig taenia coli

To prepare single smooth muscle cells from the taenia coli of guinea pig, the application of papain to the enzymatic solution was examined under two conditions: 1) the isolation in a modified Tyrode solution (containing 0.18 mM Ca2+: 0.18 mM Ca2+-Tyrode solution) and 2) the isolation in a high-K+ Tyrode solution (Na+ was replaced by K+, and Ca2+ was not added: high-K+ Tyrode solution). The presence of papain during collagenase digestion reduced contamination of broken cells and cell debris. In the case of the high-K+ Tyrode solution, papain increased the yield of single cells significantly. The cells were contracted in a dose-dependent manner by Ca2+ in the high-K+ Tyrode solution and by carbachol in 0.18 mM Ca2+-Tyrode solution; furthermore, the contractions were antagonized by verapamil and atropine, respectively. Treatment with papain did not affect cell sensitivity to the stimulants. Therefore, our results suggest that the addition of papain is useful for the isolation of single cells to investigate the physiological and pharmacological characteristics of smooth muscle.     

Isolation,culture, and characterization of smooth muscle cells from human intracranial aneurysms

Background  

Smooth muscle cells (SMCs) play a critical role in the vascular wall and also participate in vascular repair mechanisms. Dysfunction of SMCs may also contribute to the formation of intracranial aneurysms (IAs) causing subarachnoid hemorrhage. Our aim was to investigate the possibility of using cultured SMCs as an in vitro model for the study of aneurysmal SMCs.     

One-stage operation of ureteropelvic junction and ureterovesical junction stricture

One-stage surgical management for ureteropelvic (UP) and ureterovesical (UV) strictures is reported. A 3-year-old boy with UP and UV strictures on left side, underwent nephrostomy at 4 months old. The plastic operations which were Anderson-Hynes method for UP stricture and submucosal tunnel method with tailoring of dilated ureter for UV stricture were performed at the same time. The post-operative intravesical pyelogram and renal scintigraphy showed the improvement of hydronephrosis. Many authors have described two-stage operations for such cases. Our findings revealed that one-stage operation, carefully preserving the blood supply to the ureter, would be an alternative surgical management for UP and UV strictures.     

Determination of Ca2+-release from the isolated smooth muscle cells in suspension from the guinea-pig ileum

Collagenase-dispersed cells from the guinea-pig ileum were prepared and Ca2+ release into Ca2+-depleted solution from the isolated single cells obtained by the centrifugation of the dispersed cells on isotonic sucrose solution was determined with a Ca2+-selective electrode. A technique employing an isotonic sucrose solution for washing isolated cells permitted removal of contaminating extracellular fluids and obtaining the isolated cells with minimum loss of cellular Ca2+. The release of Ca2+ from the dispersed cells consisted of at least two phases. The Ca2+ release into an isotonic sucrose-Tris solution (ISTS) was significantly reduced and the early phase of the Ca2+ release disappeared. At 16 degrees C, Ca2+ release depended on the composition of the bathing solution in a Ca2+-free salt solution, the later phase of Ca2+-release was abolished whereas in ISTS both phases disappeared. Furthermore, Ca2+ release was significantly reduced after the treatment of the dispersed cells with disperse (1,500 units/ml) for 10 min. These results show that Ca2+ release into Ca2+-depleted solution from the isolated ileal cells with minimum loss of cellular Ca2+ show a biphasic curve and suggest that Ca2+ sources responsible for these phases may be of distinct origin.     

Kit-positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle

OBJECTIVE: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. MATERIALS AND METHODS: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. RESULTS: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). CONCLUSION: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra.     

Smooth muscle cell apoptosis and defective neural development in congenital ureteropelvic junction obstruction

PURPOSE: We assessed the smooth muscle cell apoptosis along with changes in cellular and extracellular components of the ureteropelvic junction in 23 patients with unilateral obstruction and compared them with 25 autopsies from ureteropelvic junction regions of age matched cadavers. MATERIALS AND METHODS: Tissue specimens obtained from pyeloplasty were divided into 3 sections-renal pelvis above the obstruction, obstructed ureteropelvic junction and ureter below the obstructed region. For the control group the normal ureteropelvic junctions of age matched infants were autopsied. In paraffin embedded sections we determined myocyte apoptosis index (using TUNEL assay), and the amount of muscular components and nerve terminals (using image analysis techniques after immunohistochemical staining). The collagen and elastin fibers were specifically stained for evaluation of changes in extracellular matrix. RESULTS: Smooth muscle cell apoptosis index was significantly increased at the site of ureteropelvic junction obstruction (5.68 +/- 0.18) compared to normal autopsied ureteropelvic junctions (3.60 +/- 0.11) and 2 other sections of obstructed ureteropelvic junction complex (renal pelvis 4.73 +/- 0.16, and ureter 3.97 +/- 0.16). The number of nerve terminals and the percentage of muscular component were significantly lower at the obstructed segments of affected patients compared to normal ureteropelvic junctions. Meanwhile, collagen fibers formed a significantly higher proportion of ureteral wall at the site of obstruction. Interestingly, there was negative correlation between myocyte apoptosis indices and number of nerve endings as well as amount of muscular components at the site of ureteropelvic junction obstruction. However, positive correlations were found between smooth muscle cell apoptosis and the percentage of collagen and elastin fibers. CONCLUSIONS: Our findings suggest an important role for myocyte apoptosis and defective neural development in the pathogenesis of congenital ureteropelvic junction obstruction that could pave the road for the emergence of new therapeutic modalities.     

The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells

PURPOSE: We generated and characterized a convenient isolated cell model of human detrusor smooth muscle to understand mechanisms that may underlie detrusor instability and provide a suitable model to test potentially useful drugs. MATERIALS AND METHODS: The electrophysiological properties of freshly isolated detrusor smooth muscle cells from human and guinea pig biopsies were compared with those undergoing cell culture to document in detail the changes that occur during primary culture and subsequent passages as well as the differences in the 2 species. RESULTS: Resting electrical characteristics were changed in the cultured cells. Membrane potential was less negative (guinea pig -59 versus -42 mV.) and membrane resistance was less (138 versus 124.5 Omegacm.(2)). Regenerative action potentials were recorded in cultured and freshly isolated cells. In guinea pig cells the overall duration and initial rate of depolarization (upstroke) was slower in cultured than in freshly isolated cells, indicative of a decreased magnitude of ionic current in cultured cells. Human cells had a similar prolongation in culture but no decrease in the upstroke rate. Experiments with selective blockers indicated that depolarization is due to influx through L-type Ca2+ channels and repolarization occurred via Ca2+ dependent K+ channels in freshly isolated and cultured cells. No further changes to properties were observed in cells passaged up to 3 times from primary cultured cells. CONCLUSIONS: Cell culture qualitatively preserves the electrophysiological properties of detrusor smooth muscle cells, although there is some decrease in channel density.     

肌源性干细胞分离培养及诱导分化为平滑肌细胞的研究

目的探讨兔肌源性干细胞分离、鉴定及诱导分化为平滑肌细胞的方法。方法取1．5个月龄新西兰兔大腿肌肉，采用Preplate技术分离培养肌源性细胞，并进行流式细胞仪（FCM）、免疫细胞化学、Western blot等确定细胞表型。采用添加血管内皮生长因子（VEGF）和与兔阴茎海绵体平滑肌细胞（CCSMC）共培养的方法诱导分离而得的细胞分化为平滑肌细胞并运用免疫细胞化学和Western blot检测特异性α-平滑肌肌动蛋白（α—SMA）的表达。结果经过连续6d的差速贴壁分离得小圆形或短梭形的小体积细胞，FCM结果显示这些细胞〉80％为desmin＋，〉70％为bcl-2^＋，〉95％为CD45^-。免疫细胞化学定性结果显示desmin＋。高汇合度（〉50％）或低血清培养时容易融合形成肌管或肌细胞核链。诱导处理后分离的细胞表达α—SMA。结论采用Preplate技术能很好地分离出肌源性干细胞，并能在体外诱导分化为平滑肌细胞，为组织工程提供种子细胞。     

Patch grafting the renal pelvis and ureteropelvic junction

Summary Lyophilized dura mater (Lyodura) and autogenous free peritoneum were compared in the replacement of the partially resected renal pelvis and ureteropelvic junction (UPJ) in the pig. The Lyodura and peritoneum grafts were both progressively resorbed, and replaced by fibroblasts which formed a mature scar lined by transitional epithelium, but without smooth muscle regeneration. Lyodura was found to be superior to free peritoneum with a longer time before resorption of the graft, less intense inflammatory reaction, absence of metaplastic bone formation, less risk of urine leak and greater ease of surgical manipulation.Supported by the University Urological Research Foundation