Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

The development of the bladder trigone, the center of the anti-reflux mechanism

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development.     

Muscular tunic of the dog ureter in the normal state and in ureterohydronephrosis (data of scanning electron microscopy)

By means of scanning electron microscopy and macro- microscopical methods in 16 mongrel dogs the ureters have been studied, normal and at ureterohydronephrosis. The ureteral muscles are spirally shaped. The external muscular layer is oriented along the spiral, its step approximately corresponds to the length of the peristaltic wave. The spiral-shaped course of the muscular fasciculi in the middle layer is of opposite direction, the spiral step corresponds to the ureter diameter. The internal muscular layer is formed as a result of changes in the spiralization angle of the middle muscular layer from outside into inside. Fibrills of the connective tissue framework possess predominantly longitudinal orientation. At ureterohydronephrosis, together with increasing diameter of the ureter, the step of the muscular spiral decreases, the connective tissue fibrillar framework acquires a net-like structure.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Smad4 Regulates Ureteral Smooth Muscle Cell Differentiation during Mouse Embryogenesis

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.     

The inhibitory effects of prostaglandin E1 on guinea-pig ureter.

Prostaglandin E1 (PGE1) hyperpolarized the smooth muscle cells of guinea-pig ureter in normal Krebs solution and was without effect on ureters depolarized in KCl Krebs, PGE1 inhibited both electrically induced contractions and K+-induced contractures of the ureters. Conditions that favored greater tension development by the ureters, namely, high [K+] or high [Ca-2+] reduced the inhibitory effects of PGE1 on the K+-induced contractures. Depolarization of guinea-pig ureter with KCl Krebs led to an increase in radio-calcium content of the tissue over a 30 min loading period. This increase in the tissue's radio-calcium content was further increased by PGE1 but not by theophylline, PGE1 was found to have no effect on either total calcium content or the calcium efflux from the tissue. It is suggested that PGE1 exerts its inhibitory action by increasing calcium sequestration at the inner surface of the cell membrane.     

A theoretical study of the effect of airway smooth muscle orientation on bronchoconstriction

If airway smooth muscle shortened in vivo to the extent that it does in vitro, then maximal bronchoconstriction would result in complete closure of virtually all airways. The fact that this does not happen indicates the existence of inhibitory mechanisms preventing maximal muscle shortening. There are many factors potentially limiting shortening in vivo. In this study we investigated one of these factors, the orientation of the smooth muscle around the airway wall. The airway was modeled as a cylinder of given wall thickness around which the muscle was wound as a spiral. The longitudinal and circumferential elasticities of the airway were embodied in a 2 x 2 matrix of elastic coefficients. We investigated smooth muscle shortening under three conditions: 1) a longitudinally stiff airway, 2) a circumferentially stiff airway, and 3) a longitudinally and circumferentially compressible airway. In case 1, for a given degree of smooth muscle shortening, airway resistance increased markedly with increasing pitch of the smooth muscle spiral. On the other hand, the muscle tension required to elicit a given change in resistance also increased markedly with pitch. In case 2, the effect with increasing pitch was reversed. In case 3, resistance first increased and then decreased as spiral pitch increased. Similarly, the muscle tension required to elicit a given change in resistance first increased and then decreased with pitch. These results suggest that the orientation of the smooth muscle about the airway may be very important in determining airway responsiveness.     

基因谱系示踪揭示小鼠Tbx18+肾脏间质祖细胞分化为输尿管平滑肌

本文旨在研究Tbx18+肾脏间质祖细胞分化为输尿管平滑肌细胞的命运及转录因子Tbx18在小鼠输尿管平滑肌发育形成中起到的作用．实验建立Tbx18:Cre/R26REYFP和Tbx18:Cre/R26RLacZ两种谱系示踪系统和Tbx18:Cre/Cre 敲除模型．该示踪模型通过cre重组酶的表达能有效地示踪Tbx18+肾脏间质祖细胞在泌尿系统的发育命运．通过免疫荧光染色和X-gal染色,同时发现Tbx18+肾脏间质祖细胞可分化为输尿管平滑肌细胞,但不分化为输尿管移行上皮细胞．在Tbx18:Cre/Cre基因突变模型中,泌尿系统出现明显的肾积水和输尿管积水,肾盏、肾盂扩张,输尿管明显缩短和扩张．实验结果揭示,Tbx18+ 肾脏间质祖细胞可以分化为输尿管平滑肌细胞,且转录因子Tbx18在哺乳动物输尿管平滑肌的发育中起到重要的作用．     

Local longitudinal muscle shortening of the human esophagus from high-frequency ultrasonography

We analyzed local longitudinal shortening by combining concurrent ultrasonography and manometry with basic principles of mechanics. We applied the law of mass conservation to quantify local axial shortening of the esophageal wall from ultrasonically measured cross-sectional area concurrently with measured intraluminal pressure, from which correlations between local contraction of longitudinal and circular muscle are inferred. Two clear phases of local longitudinal shortening were observed during bolus transport. During luminal filling by bolus fluid, the muscle layer distends and the muscle thickness decreases in the absence of circular or longitudinal muscle contraction. This is followed by local contraction, first in longitudinal muscle, then in circular muscle. Maximal longitudinal shortening occurs nearly coincidently with peak intraluminal pressure. Longitudinal muscle contraction begins before and ends after circular muscle contraction. Larger longitudinal shortening is correlated with higher pressure amplitude, suggesting that circumferential contractile forces are enhanced by longitudinal muscle shortening. We conclude that a peristaltic wave of longitudinal muscle contraction envelops the wave of circular muscle contraction as it passes through the middle esophagus, with peak longitudinal contraction aligned with peak circular muscular contraction. Our results suggest that the coordination of the two waves may be a physiological response to the mechanical influence of longitudinal shortening, which increases contractile force while reducing average muscle fiber tension by increasing circular muscle fiber density locally near the bolus tail.     

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.     

Further observations on upper urinary tract smooth muscle

Summary Light and electron microscopic techniques have been employed to study the arrangement and distribution of two types of muscle in the upper urinary tract of the rat. An outer layer of cells has been identified in the wall of the renal calix and pelvis. These cells are separated by connective tissue but possess numerous processes which make close contacts with adjacent cells. A layer of similar cells has not been observed in the wall of the upper ureter. The inner layer of muscle in the calix and pelvis is composed of larger cells similar to and apparently continuous with ureteric muscle. These cells are closely related to one another without intervening connective tissue and possess numerous bundles of myofilaments which extend along the length of the cell. The two types of muscle are closely related and, in the junctional region, cells of the outer layer are arranged along the length and make close contacts with one or more of the inner smooth muscle cells. A quantitative estimation has been made of nerve bundles associated with smooth muscle forming the outer layer of the calix and pelvis and with the muscle of the ureter. The results have shown a five fold increase in nerves associated with the caliceal muscle when compared with the ureter. The results are discussed in relation to the concept of a ureteric pacemaker.The authors wish to thank Professor G. A. G. Mitchell for his useful advice and encouragement.     

Luminal morphology of small arterial vessels in the contracted spleen,studied by scanning electron microscopy of microcorrosion casts

Unique luminal configurations exhibited by small arterial vessels in contracted spleens of dog and cat were studied by means of vascular corrosion casts examined by scanning electron microscopy. Concertina-like pleating was seen in casts of trabecular arteries/arterioles, whereas within lymphatic nodules arteriolar casts lacked pleating and were smooth and uniformly cylindrical (as were all small arterial vessels in distended spleens). Morphological details of arterial vessels observed in histological sections indicated that pleating is not due to contraction of specially arranged vascular smooth muscle but to overall shortening of trabecular arterial vessels, caused by contraction of longitudinal smooth muscle in trabeculae. Another phenomenon observed in casts from contracted spleens was an almost complete "pinching-off" of many arteriolar lumens; histological evidence indicated that this is due to contraction of vascular smooth muscle, which selectively diverts flow away from certain regions of the organ. Also noted was a markedly convoluted, tortuous configuration of arterioles (penicilli) in the red pulp of contracted spleens.     

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (～250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ～70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.     

The mechanical advantage of local longitudinal shortening on peristaltic transport

Whereas bolus transport along the esophagus results from peristaltic contractions of the circular muscle layer, it has been suggested that local shortening of the longitudinal muscle layer concentrates circular muscle fibers in the region where the highest contractile pressures are required. Here we analyze the mechanical consequences of local longitudinal shortening (LLS) through a mathematical model based on lubrication theory. We find that local pressure and shear stress in the contraction zone are greatly reduced by the existence of LLS. In consequence, peak contractile pressure is reduced by nearly 2/3 at physiological LLS, and this reduction is greatest when peak in LLS is well aligned with peak contractile pressure. We conclude that a peristaltic wave of local longitudinal muscle contraction coordinated with the circular muscle contraction wave has both a great physiological advantage (concentrating circular muscle fibers), and a great mechanical advantage (reducing the level of contractile force required to transport the bolus), which combine to greatly reduce circular muscle tone during esophageal peristalsis.     

Active contractions of ureteral segments

The first step in the analysis of the biomechanics of any organ is to obtain its constitutive equation. In pursuit of a constitutive equation describing the peristalsis of the ureter, we measured the relationship between the length of the muscle, the velocity of contraction, and the active tension development of isolated ureter segments. The results of length-tension measurements (giving the maximum tension developed in isometric contraction of a ureter segment of specific length) were similar to those obtained by previous investigators and reflected the behavior of length-tension relationship for other smooth muscles. Two aspects of the force-velocity relationship of the ureter were examined: the effect of releasing the ureter at different times after stimulation, and that at different levels of afterload. Measurements were analyzed using the hyperbolic Hill's equation in the form T/T0 = (1-v/v0) (l + cv/v0)-1 where v is the velocity of contraction, v0 is the velocity of contraction when T = 0, T is the tension in the muscle after release, T0 is the tension in the muscle immediately prior to release, and c is the dimensionless constant. The results of force-velocity measurements showed that the so-called "maximum" velocity v0, is the largest if the tension is released at a time of contraction, early in the rise portion of the contraction cycle. Further, if tension is released from an isometric contraction at a fixed time in the rise portion of the contraction cycle, the largest value of v0 is obtained when the muscle length is in the range of 0.85-0.90 Lmax. Interestingly, the in vivo length of the ureter lies also in this range, 0.85-0.90 Lmax.     

Oesophageal peristalsis in the cat: the role of central innervation assessed by transient vagal blockade

Studies were performed on five cats to assess the role of extrinsic vagal innervation in the control of peristalsis in the smooth muscle oesophagus. Transient vagal nerve blockade was accomplished by cooling the cervical vagosympathetic nerve trunks previously isolated in skin loops on each side of the neck. Peristalsis throughout the body of the oesophagus was monitored using a continuously perfused multilumen manometry tube. Striated and smooth muscle portions of the esophagus were delineated by abolishing smooth muscle activity with atropine. Secondary peristalsis was assessed by intra-oesophageal balloon distension studies. The threshold volume for balloon-induced secondary peristalsis was lower in the smooth muscle oesophagus. Unilateral vagal blockade reduced the incidence of primary and secondary peristalsis in the striated muscle oesophagus but not in the smooth muscle oesophagus. Bilateral vagal nerve blockade abolished primary swallow-induced peristalsis and secondary peristalsis in both the smooth and striated muscle cat oesophagus. Administration of cholinergic agents or adrenergic blocking agents failed to restore secondary peristalsis in the smooth muscle oesophagus during vagal cooling. We conclude that connections to the central nervous system via the vagal nerve trunks are required for normal secondary as well as primary peristalsis in both the smooth and striated muscle portions of the cat oesophagus.     

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo‐4, Propidium iodide, and di‐8‐Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin‐eosin, Masson's tri‐chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

Airway remodeling in asthma amplifies heterogeneities in smooth muscle shortening causing hyperresponsiveness.

Although airway remodeling and inflammation in asthma can amplify the constriction response of a single airway, their influence on the structural changes in the whole airway network is unknown. We present a morphometric model of the human lung that incorporates cross-sectional wall areas corresponding to the adventitia, airway smooth muscle (ASM), and mucosa for healthy and mildly and severely asthmatic airways and the influence of parenchymal tethering. A heterogeneous ASM percent shortening stimulus is imposed, causing distinct constriction patterns for healthy and asthmatic airways. We calculate lung resistance and elastance from 0.1 to 5 Hz. We show that, for a given ASM stimulus, the distribution of wall area in asthmatic subjects will amplify not only the mean but the heterogeneity of constriction in the lung periphery. Moreover, heterogeneous ASM shortening that would produce only mild changes in the healthy lung can cause hyperresponsive changes in lung resistance and elastance at typical breathing rates in the asthmatic lung, even with relatively small increases in airway resistance. This condition arises when airway closures occur randomly in the lung periphery. We suggest that heterogeneity is a crucial determinant of hyperresponsiveness in asthma and that acute asthma is more a consequence of extensive airway wall inflammation and remodeling, predisposing the lung to produce an acute pattern of heterogeneous constriction.