Information

Why did the urinary bladder evolve?


Sure it's convenient to decide when to urinate but not essential for survival or reproduction, as I understand. But just convenience is not a drive for evolution.

Does the bladder serve any essential purpose? If not why did bladders evolve?


Here are just a few points that might apply:

  • Urine is used for scent marking by some species, so the ability to store urine could be useful.
  • At the opposite side, controlling the release of a strong scent would help in stealth for both predators and prey. (In addition, a single strong scent might temporarily overload a predator's sense of smell making tracking more subtle scents more difficult.)
  • Flushing an excretion point (single point reduces opportunities for invasion) under some pressure could help avoid blockage and parasitic invasion/accumulation. (Providing a tube from the extraction organ to the excretion point allows more flexible (and protected) placement of the organ, but also increasing the benefit of a flushing mechanism.)
  • Flushing could also reduce contact with skin. Urine might act as an irritant and a nutrient source for parasites.
  • Adding a buffer is a common technique for any pipelined operation to allow smaller resources to handle temporal variation in input and output rate. Without a such a buffer, all stages have to be sized for the maximum utilization rather than something closer to average utilization.
  • Avoiding potential contamination of food and water may also be a benefit of controlled urination (or excreting might have a fertilizing or pest-deterrent aspect for plants).

Since terrestrial animals presumably retained urinary bladders developed by their marine ancestors, benefits associated with terrestrial lifestyle would only provide selective pressure to retain such a feature. However, if somehow a line of terrestrial animals abandoned urinary bladders, it is not entirely implausible that even scent marking benefits could increase the selective pressure enough to overcome some peculiar opposing selective pressure.

Initially, the animal might apply scents by scratching at an area of skin irritated by urine release against some surface. Then this scratching might be preferentially located (i.e., a scent marking behavior is developed). Having such a behavior would then obvious bring benefits to storing a significant amount of urine and eventually to being able to squirt the urine (allowing the scratching behavior to fall away).

The above are just somewhat reasonable speculations about what selective pressures might encourage the development (and retention) of a urinary bladder. Hopefully, someone with actual knowledge will provide a better answer documenting established theory and evidence for how urinary bladders actually developed.


A few observations to add to Paul's:

Urinating on yourself in winter could be a fatal thermoregulatory mistake.

Urinating on your substrate could hinder locomotion.

Having a continual slick of high osmolarity fluid on your skin would be damaging to the epithelium and work at odds to the action of the kidneys.


Apparently, the urinary bladder is not unique to mammals, and is even found in fish, which one might presume can urinate at any time or even better - expel ammonia out of their gills, so why do they need a bladder?

I found the following article trying to understand what the bladder does in freshwater fish: https://jeb.biologists.org/content/155/1/567.

Apparently they concluded that the bladder is important because the kidneys let too much useful stuff (like Na and Cl ions) out, and letting it sit in the bladder for 30 minutes (as measured in the trouts in that research) allows some of these ions to be re-absorbed by the cells lining the bladder. I don't know if this is why the bladder evolved (which would have been easy - just as an enlarged cavity in front of the kidney holding some urine before it is expelled), or why couldn't a better kidney - absorbing more salts - have evolved instead. I also don't know if mammals still use the urinary bladder in this way. But once it has evolved, it is here to stay, unless it has a negative survival contribution. And as other answers suggested, it probably has positive contribution, if anything - such as avoiding leaving a strong trail of scent leading to you, or avoiding soiling your den with urine.


Voiding Function and Dysfunction Urinary Incontinence

Alan J. Wein MD, PhD , M. Louis Moy , in Penn Clinical Manual of Urology , 2007

IV OVERVIEW OF THE MICTURITION CYCLE: SIMPLIFICATION

Bladder accommodation during filling is a primarily passive phenomenon. It is dependent on the elastic and viscoelastic properties of the bladder wall and the lack of parasympathetic excitatory input. An increase in outlet resistance occurs via the striated sphincter somatic guarding reflex. In at least some species a sympathetic reflex also contributes to storage by (1) increasing outlet resistance by increasing tension on the smooth sphincter, (2) inhibiting bladder contractility through an inhibitory effect on parasympathetic ganglia, and (3) causing a decrease in tension of bladder body smooth muscle. Continence is maintained during increases in intra-abdominal pressure by the intrinsic competence of the bladder outlet and the pressure transmission ratio to this area with respect to the intravesical contents. A further increase in striated sphincter activity, on a reflex basis, is also contributory.

Emptying (voiding) can be voluntary or involuntary and involves an inhibition of the spinal somatic and sympathetic reflexes and activation of the vesical parasympathetic pathways, the organizational center for which is in the brain stem. Initially, there is a relaxation of the outlet musculature, mediated not only by the cessation of the somatic and sympathetic spinal reflexes but probably also by a relaxing factor, very possibly nitric oxide, released by parasympathetic stimulation or by some effect of bladder smooth muscle contraction itself.

A highly coordinated parasympathetically induced contraction of the bulk of the bladder smooth musculature occurs, with shaping or funneling of the relaxed outlet, due at least in part to a smooth muscle continuity between the bladder base and proximal urethra. With amplification and facilitation of the bladder contraction from other peripheral reflexes and from spinal cord supraspinal sources, and the absence of anatomic obstruction between the bladder and the urethral meatus, complete emptying will occur.

Whatever disagreements exist regarding the anatomic, morphologic, physiologic, pharmacologic, and mechanical details involved in both the storage and expulsion of urine by the LUT, we believe that agreement is found regarding certain points. First, the micturition cycle involves two relatively discrete processes: bladder filling /urine storage and bladder emptying/voiding. Second, whatever the details involved, these processes can be summarized succinctly from a conceptual point of view.

Bladder filling/urine storage require the following:

Accommodation of increasing volumes of urine at a low intravesical pressure (normal compliance) and with appropriate sensation.

A bladder outlet that is closed at rest and remains so during increases in intra-abdominal pressure.

Absence of involuntary bladder contractions (detrusor overactivity [DO]).

Bladder emptying/voiding require the following:

A coordinated contraction of the bladder smooth musculature of adequate magnitude and duration.

A concomitant lowering of resistance at the level of the smooth and striated sphincter.

Absence of anatomic (as opposed to functional) obstruction.

Any type of voiding dysfunction must result from an abnormality of one or more of the factors previously listed regardless of the exact pathophysiology involved. This division, with its implied subdivision under each category into causes related to the bladder and the outlet, provides a logical rationale for discussion and classification of all types of voiding dysfunction and disorders as related primarily to bladder filling/urine storage or to bladder emptying/voiding. There are some types of voiding dysfunction that represent combinations of filling and storage and emptying and voiding abnormalities. Within this scheme, however, these become readily understandable, and their detection and treatment can be logically described. Further, using this scheme, all aspects of urodynamic, radiologic, and video urodynamic evaluation can be conceptualized as to exactly what they evaluate in terms of either bladder or outlet activity during filling and storage or emptying and voiding. Treatments for voiding dysfunction can be classified under broad categories according to whether they facilitate filling and storage or emptying and voiding and whether they do so by acting primarily on the bladder or on one or more of the components of the bladder outlet. Finally, the individual disorders produced by various neuromuscular dysfunctions can be considered in terms of whether they produce primarily storage or emptying abnormalities or a combination.


Introduction

The urinary bladder in mammals and other terrestrial animals is a muscular distensible organ that holds urine under low pressure and can be emptied under voluntary control. How can this organ evolve from a thin-walled structure in fish that is involved with electrolyte and water exchange? Teleost fish have a confluence of their ureters that forms a thin-walled urinary bladder. In salt water, fish lose most (90%) of their nitrogenous waste via their gills [1]. In fresh water, there is a demand for more urinary excretion of nitrogenous waste. Under brackish water conditions, fish that survive have even less ability to exchange ammonia across their gills, and excretion via the kidneys and urinary tract becomes a greater importance.

Teleost fish have a pronephros and mesonephros, but no metanephros. The electrolyte reabsorption and secretion and water reabsorption are limited compared to animals that have a metanephros and loop of Henle. The lower urinary tract and bladder in fresh water fish is a thin-walled organ that is involved in electrolyte exchange, not a storage organ. For example, rainbow trout bladders are capable of water and sodium reabsorption [2]. Mudskippers (amphibious gobioids, e.g. Periophthalminae of the family Oxudercinae) are a living example of amphibious fish that can spend several days out of water. They may represent an example of co-evolution that might be similar to the first amphibious animals to attempt to live on land, but are not living fossils [3] . They also might represent a morphological model for the ancestors of the first tetrapods. The mudskipper is even capable of producing urea when air-breathing [4].

Urinary bladders are found in many different animals and, it has been suggested, may have evolved twice [5].

There is an evolutionary argument for the development of a storage function in the urinary bladder that has been suggested by several authors [5] . If an animal crawled onto land and left a continuous scent trail, then this could be easily followed by a predator. By storing urine and discretely passing it in an intermittent fashion, the scent trail would be harder to follow, and the animal (prey) has a selective survival advantage.

Agent-based modelling is a way of describing emergent phenomena based on individual components of a complex system. An early example of this is Thomas Schelling’s ‘Dynamic Models of Segregation’ [6]. These techniques have then been used in areas such as population dynamics, for example ‘Aphid Population Dynamics of Agricultural Landscapes: An Agent-Based Simulation Model’ [7]. More recently, agent-based modelling has been used to describe the emergence of the evolution of grammar by Luc Steels [8].

In this study, agent-based modelling was used in order to test the hypothesis that there is a selective advantage to the evolution of a bladder with urinary storage capacity in prey. By considering continuous and discontinuous scent trails of individual prey and predators (the agents) in random walks on a two-dimensional grid, hunts were simulated in two phases: detection and pursuit. That is, the scent marker is left by the prey first on every location it visits—continuous then with a defined separation between the prey leaving a scent marker—discontinuous. The first simulation (Sect. 2.1.1 ‘Detection of Prey’) was a simple model where the prey and predator each had a random walk and if the predators walk intercepted the preys walk, then the prey was detected. The duration of prey survival was taken to be the number of steps that the agent had taken before being detected. In the second simulation (Sect. 2.1.2 ‘Pursuit of Prey’), a Monté Carlo simulation [9] was run to model the pursuit phase of the hunt. This was achieved by using the diffusion equation in order to model scent propagation and therefore define the pungency of a scent marker, and combining this with the distance between the predator and prey a probability was worked out that the prey would be caught. Then, if a random number is generated that is greater than this probability, the prey has been caught. This was used to give simulation 1 as an approximate minimum bound for the selective advantage of having a urinary bladder with storage capacity, simplifying the methodology of the third simulation (2.1.3 ‘Evolution’). Using the detection of the prey, as in simulation 1, as a metric for the hunt, simulation 3 had 100 prey in each generation. Each prey was assigned a probability of leaving a scent marker on each step. The top ten longest surviving prey were then used to infer the probability of leaving a scent marker for the next generation, with the top survivor and bottom survivors probabilities randomly mutated either up or down.

Simulation 1 demonstrated a huge survival advantage in a discontinuous scent trail (Sect. 3.1). Simulation 2 displayed that having a discontinuous scent trail greatly magnified this advantage during the pursuit phase (Sect. 3.2). This resulted in the use of simulation 1 as a minimum bound of the survival of the prey agents in simulation 3. In simulation 3, survival of the fittest prey related strongly to inheritance of the probability of leaving a discontinuous scent trail (bladder storage capacity). Over a thousand generations, the random mutation of only two of the prey was coupled with the selective pressure of predation, was enough to give the emergence of storage capacity in the urinary bladder (Sect. 3.3). This is discussed further in Sect. 3, before concluding in Sect. 4 that there is a huge in-silico selective advantage that in this agent-based model, there is a huge selective advantage to the evolution of urinary bladder storage capacity.


How did the process of metamorphosis evolve in creatures?

All I can tell you is that it is unknown exactly and debated among biologists. One theory I read that stuck with me was that it may have begun as an advantage to hatch from your egg prematurely, possibly by shortening the window of vulnerability and/or providing the larvae with easy access to rich nutrients. A mechanism evolved to delay certain developmental events until long after hatching. Certainly one of the most fascinating examples of evolution in my opinion!

One crazy hypothesis I heard was that metamorphosis could result from distant hybridizations, where taxa across class or even phylum hybridize, resulting in bizzare body plan combinations.

This is not supported by any genetic evidence that I know of, which is kind of sad. itɽ have been crazy and cool if it were true.

I'll second the "mobile egg" as one explanation for insect metamorphosis, I've read that too.

Tadpoles are actually not dissimilar to lungfish larvae. Having a similar larval form is something they probably inherited from their fishy ancestors.

Lots of marine invertebrates have incredibly bizzare larvae. The weirdest in my opinion is sea urchin larvae. The adult form develops as a sort of bud called a rudiment on the side of the larvae. It's got a body axis at right angle to the larvae. Eventually it just sort of . drops off.

No, I really have no clue how this evolved. Echinoderms are some of the weirdest things on the planet


RESULTS

In newborn mouse urogenital tracts, the bladder is encircled by a thick layer of muscle called the detrusor and the ureters enter the trigone at the base of the bladder between the bladder and urethra(Fig. 1A,C,D). The trigone can be visualized in dissected urogenital tracts as a smooth triangular shaped region bounded by the ureters laterally, terminating at the bladder neck where the urethra begins (Fig. 1D,E). The surface of the urethra and ureters, like the bladder, is covered by the urothelium, a specialized transitional epithelium that prevents leakage and damage (Fig. 1D, the urothelium is red). The intramural ureters pass through the bladder muscle and submucosa and open into the trigone at its lateral edges(Fig. 1E). Higher magnification reveals the eyelet-shaped ureter orifice opening into the urothelium(Fig. 1F). Unlike the bladder,which is covered by folds, the trigone is generally smooth, which has led to the suggestion that its origin might be distinct from the bladder.

Development of the trigone

The trigone has been defined in a number of ways here, we will consider the trigone to be the muscular triangle bounded laterally by the ureter orifices extending posteriorly to the urethra(Fig. 1C). The unique features of the trigone including its appearance and physiological properties have led to the idea that the trigone originates from non-urogenital sinus tissue, in particular from the common nephric duct that is the caudal-most segment of Wolffian duct. However, our previous studies suggest that this is not the case because the common nephric duct undergoes apoptosis during ureter transposition, hence the trigone is likely to form in a different manner than previously thought. Other studies suggest that the trigone is formed in large part from ureteral fibers that fan out laterally forming an inter-ureteric ridge and posteriorly forming Bell's muscle(Fig. 1C). To begin to address this question we first established which muscles are present in the trigone by analyzing its formation in mouse urogenital tracts at different developmental and post-natal stages. At E15, analysis for expression of smooth muscle alpha actin revealed extensive smooth muscle differentiation (green) in the bladder,urethra and in the extra-vesicular ureters (the portion of ureter outside the bladder), but there was little if any detectable smooth muscle lining the intramural ureter (the portion of the ureter within the bladder) in the trigonal region (Fig. 2A).

Analysis of urogenital tracts at P0 revealed a thick smooth muscle coat surrounding the extra-vesicular ureter and a few longitudinal fibers surrounding the intramural ureter extending through the detrusor and submucosa(Fig. 2B,E,F). Analysis at adult stages revealed additional smooth muscle lining the intramural ureter. The trigone appeared at this stage to be a hybrid between the bladder and urethra. Its surface was smooth and free of folds like the urethra was covered by a thick muscularis submucosa, similar to that in the bladder(Fig. 2C,D,G,H). The ureteral wall outside the bladder is thick, containing at least three layers of circular and longitudinal muscle (Fig. 2E). However, as reported by other groups(Yucel and Baskin, 2004), only a small subset of longitudinal ureteral fibers extend into the intramural region, where they appear to intercalate with the bladder muscle and terminate in the submucosa, below the urothelium(Fig. 2F,G). These findings suggest that two major muscle types are present in the trigone: the bladder muscle (detrusor) and the muscle associated with the intramural ureter. Extensive analysis of whole-mount urogenital tracts, cryosections and vibratome sections did not reveal additional muscle groups reported to be part of the trigone, including an intra-ureteric bar which is said to extend laterally between the two ureter orifices, and Bell's muscle which is said to extend caudally from the ureter orifices to the trigone apex(Tanagho et al., 1968).

The trigone is the site of the anti-reflux mechanism. (A). Schematic of the trigone at the bladder base and its connections with the ureters showing the intramural ureter segment that is normally compressed to prevent back-flow of urine to the ureters and kidneys. (B) Schematic showing compression of the intramural ureter. (C) A detailed representation of the trigone, which is thought to be composed of ureteral fibers that enter the bladder via Waldeyer's sheath, fan out across the base to form the inter-ureteric ridge and extend down toward the apex to form Bell's muscle. (D) A vibratome section from an adult mouse stained for uroplakin (red) to reveal the urothelium, and for smooth muscle alpha actin(green) to reveal smooth muscle. (E) Opened bladder showing the trigone in an adult Hoxb7-Gfp mouse. The ureter orifices (yellow) are located at the base of the trigone. (F) High magnification of the ureter orifice, showing its eyelet shape at the point it opens into the urothelium(red, uroplakin). Magnification: ×100 in D,E ×200 in F.

The trigone is the site of the anti-reflux mechanism. (A). Schematic of the trigone at the bladder base and its connections with the ureters showing the intramural ureter segment that is normally compressed to prevent back-flow of urine to the ureters and kidneys. (B) Schematic showing compression of the intramural ureter. (C) A detailed representation of the trigone, which is thought to be composed of ureteral fibers that enter the bladder via Waldeyer's sheath, fan out across the base to form the inter-ureteric ridge and extend down toward the apex to form Bell's muscle. (D) A vibratome section from an adult mouse stained for uroplakin (red) to reveal the urothelium, and for smooth muscle alpha actin(green) to reveal smooth muscle. (E) Opened bladder showing the trigone in an adult Hoxb7-Gfp mouse. The ureter orifices (yellow) are located at the base of the trigone. (F) High magnification of the ureter orifice, showing its eyelet shape at the point it opens into the urothelium(red, uroplakin). Magnification: ×100 in D,E ×200 in F.

Development of the trigone. (A) Brightfield/darkfield composite showing a frontal section through an E15 embryo stained for uroplakin (red) to reveal the urothelium, and smooth muscle alpha actin(green) to reveal smooth muscle. Note the absence of muscle surrounding the intramural ureter compared with the extra-mural ureter, which already has a thick smooth coat. (B) The trigone in a newborn mouse showing the intramural ureter crossing the bladder muscle and submucosa. Note the longitudinal muscle fibers surrounding the intramural ureter. (C) The trigone in an adult mouse. (D) The bladder of a newborn mouse showing the deep folds of the lining, and the muscularis mucosa and smooth muscle layers below. (E) Higher magnification of the ureteral tunnel shown in B. (F) High-magnification image of the intramural ureter showing the longitudinal muscle fibers (green). (G) Higher magnification of the region in C showing the position in the trigone where the ureter joins. Note the longitudinal fibers that intercalate with the bladder muscle (yellow arrows). (H) The urethra in a newborn mouse showing the thick muscle coat (green) and smooth urothelial surface (red). Magnification: ×50 in A-C ×100 in D,E,G,H ×200 in F.

Development of the trigone. (A) Brightfield/darkfield composite showing a frontal section through an E15 embryo stained for uroplakin (red) to reveal the urothelium, and smooth muscle alpha actin(green) to reveal smooth muscle. Note the absence of muscle surrounding the intramural ureter compared with the extra-mural ureter, which already has a thick smooth coat. (B) The trigone in a newborn mouse showing the intramural ureter crossing the bladder muscle and submucosa. Note the longitudinal muscle fibers surrounding the intramural ureter. (C) The trigone in an adult mouse. (D) The bladder of a newborn mouse showing the deep folds of the lining, and the muscularis mucosa and smooth muscle layers below. (E) Higher magnification of the ureteral tunnel shown in B. (F) High-magnification image of the intramural ureter showing the longitudinal muscle fibers (green). (G) Higher magnification of the region in C showing the position in the trigone where the ureter joins. Note the longitudinal fibers that intercalate with the bladder muscle (yellow arrows). (H) The urethra in a newborn mouse showing the thick muscle coat (green) and smooth urothelial surface (red). Magnification: ×50 in A-C ×100 in D,E,G,H ×200 in F.

The trigone is evolutionarily conserved

The failure to identify structures in the mouse thought to be associated with the trigone suggests that either the trigone is formed differently than previously thought, or that there are substantial differences in the structure of the mouse and human trigone. To address this question, we compared the trigone in human and mouse. Sections through the trigone of a 22-week human fetus stained for smooth muscle alpha actin revealed the ureter passing through the bladder muscle and into the submucosa(Fig. 3A). The morphology of the bladder muscle, which is organized in bundles, was seen to be distinct from the thin longitudinal smooth muscle fibers that surround the ureter(Fig. 3A,C). Analysis of the mouse trigone at similar stages revealed few, if any, differences. The ureter is ensheathed in a thin layer of longitudinal smooth muscle one or two cell layers thick, surrounded by and distinct from the bladder muscle(Fig. 3B). Cross-sections through the ureter as it passes through the bladder revealed extensive similarity across species. The intramural ureter in the human trigone is surrounded by a thin layer of longitudinal fibers that are most likely ureteral smooth muscle, similar to that in the section through the mouse trigone at a comparable level (Fig. 3C,D). The observation that the mouse trigone displays similar morphology and muscle arrangement to that in human suggests that the trigone develops in a similar manner in both species, and is likely to be formed primarily from the ureter and bladder muscle.

Lineage analysis reveals the origin of trigonal muscle

Ureteral muscle is thought to make a major contribution to the trigone(Roshani et al., 1996 Tanagho et al., 1968 Woodburne, 1964). However,given the complexity of the trigonal region it is not possible to determine whether this is the case by visual inspection. To address this question, we performed lineage studies permanently labeling smooth muscle progenitors in the ureter using the Cre-lox recombination system. We then followed the fate of ureteral mesenchymal cells at late stages of development to determine whether their descendents populate the trigone. We crossed Rarb2-Cremice (Kobayashi et al., 2005),which express the Cre recombinase in mesonephric mesenchyme surrounding the nephric duct, in mesenchymal cell types within the kidney and in ureteral mesenchyme (Kobayashi et al.,2005), with Rosa26 lacZ reporter (R26RlacZ) mice(Soriano, 1999). lacZexpression is permanently activated in cells expressing both the Rosa26 reporter and the Rarb2-Cre transgene and in their descendents, enabling us to determine the contribution of ureteral muscle to the trigone.

Comparison of the trigone in humans and mice. (A) A section through the human trigone at the level of the intramural ureter stained for smooth muscle alpha actin (brown). Black arrows point to the intramural muscle fibers. (B) A section through a newborn mouse showing the trigone stained for smooth muscle alpha actin (green) and the urothelium stained for uroplakin (red). The yellow arrows point to the longitudinal ureteral muscle fibers that encircle the intramural ureter. (C) Section through a human trigone showing the intramural path of the ureter and its surrounding thin layer of fibers (black arrows). (D). Section through the mouse trigone at birth showing the path of the intramural ureter, stained for uroplakin(red) to reveal the urothelium and smooth muscle alpha actin (green). The yellow arrows point to the longitudinal muscle fibers associated with the intramural ureter. Magnification: ×20.

Comparison of the trigone in humans and mice. (A) A section through the human trigone at the level of the intramural ureter stained for smooth muscle alpha actin (brown). Black arrows point to the intramural muscle fibers. (B) A section through a newborn mouse showing the trigone stained for smooth muscle alpha actin (green) and the urothelium stained for uroplakin (red). The yellow arrows point to the longitudinal ureteral muscle fibers that encircle the intramural ureter. (C) Section through a human trigone showing the intramural path of the ureter and its surrounding thin layer of fibers (black arrows). (D). Section through the mouse trigone at birth showing the path of the intramural ureter, stained for uroplakin(red) to reveal the urothelium and smooth muscle alpha actin (green). The yellow arrows point to the longitudinal muscle fibers associated with the intramural ureter. Magnification: ×20.

Ureteral fibers contribute to the trigone. (A) Sagittal section through a Rarb2-CreR26RlacZ embryo at E14 showing lacZ-expressing mesenchymal cells surrounding the ureter (yellow arrowheads in all panels). Note the absence of lacZ-expressing cells in the bladder, trigone and urethra. (B) Higher magnification of a region of A. (C) Whole-mount of a newborn Rarb2-CreR26RlacZurogenital tract showing lacZ-expressing smooth muscle cells lining the extra-mural and intramural ureter. (D) A section through the trigone showing lacZ-expressing cells surrounding the intramural ureter. (E) Smooth muscle uroplakin staining of a section serial to D,showing that the lacZ activity in D corresponds to smooth muscle.(F). Section through a human fetus at the same level as E, showing the ureteral muscle embedded in bladder muscle in the trigone. wd Wolffian duct. Magnification: ×100 in A ×200 in B-F.

Ureteral fibers contribute to the trigone. (A) Sagittal section through a Rarb2-CreR26RlacZ embryo at E14 showing lacZ-expressing mesenchymal cells surrounding the ureter (yellow arrowheads in all panels). Note the absence of lacZ-expressing cells in the bladder, trigone and urethra. (B) Higher magnification of a region of A. (C) Whole-mount of a newborn Rarb2-CreR26RlacZurogenital tract showing lacZ-expressing smooth muscle cells lining the extra-mural and intramural ureter. (D) A section through the trigone showing lacZ-expressing cells surrounding the intramural ureter. (E) Smooth muscle uroplakin staining of a section serial to D,showing that the lacZ activity in D corresponds to smooth muscle.(F). Section through a human fetus at the same level as E, showing the ureteral muscle embedded in bladder muscle in the trigone. wd Wolffian duct. Magnification: ×100 in A ×200 in B-F.

Analysis of Rarb2-CreR26RlacZ embryos at E14 revealed lacZ expression in mesenchymal cells around the ureters, but not in smooth muscle progenitors in the bladder and trigone(Fig. 4A,B). At birth, lacZ expression persisted in smooth muscle cells in the extra-vesicular ureter coat in both circular and longitudinal fibers, which were most likely descendents of the labeled mesenchymal cells observed at E14,but not in the bladder or urethra (Fig. 4C). In the trigonal region, careful analysis revealed lacZ activity in the longitudinal fibers surrounding the ureter that extended into the bladder muscle and submucosa(Fig. 4D,E). Despite the large amount of muscle in this region, we did not observe ureteral fibers extending further into the trigone, which have been postulated to generate the inter-ureteric bar, nor into the posterior trigone extending toward the urethra, which have been postulated to form Mercier's bar(Fig. 4C,D). Comparison of the distribution of muscle in the mouse and human trigone at this stage revealed few, if any, differences (Fig. 4E,F), suggesting that the failure to identify a more extensive contribution from ureteral fibers is not due to interspecies differences. These findings suggest that the trigone is formed predominantly from bladder muscle, with a contribution from ureteral fibers that is much more limited than previously thought.

The trigone is formed predominantly from bladder muscle

Histological studies suggest that two muscle groups reside in the trigonal region: the detrusor muscle of the bladder and longitudinal ureteral fibers. To assess the contribution of bladder muscle to the trigone, we permanently labeled bladder and urethral mesenchymal muscle progenitors by crossing R26RlacZ reporter mice with a Sm22-Cre mouse line in which the Cre recombinase is expressed in urogenital sinus mesenchyme but not in ureteral mesenchyme (Kuhbandner et al.,2000) (Fig. 5). Beginning at E12, Sm22-CreR26RlacZ embryos displayed extensive lacZ activity in mesenchymal cells in the bladder, the trigone and the urethra, but not in the ureters or Wolffian ducts(Fig. 5A and data not shown). By birth, expression was throughout the muscle in the bladder, trigone and urethra, but there were few if any lacZ-labeled smooth muscle cells in the ureter, including the intramural ureter in the trigonal region(Fig. 5B,C). The distribution of lacZ activity in the trigonal region of Sm22-CreR26RlacZmice was compared with that of smooth muscle alpha actin in wild-type embryos. This revealed that there is indeed muscle present in this lateral portion of the trigone at the ureteral junction, and that these unlabeled cells are likely to correspond to ureteral muscle(Fig. 5D,E). Comparison with sections from human trigone revealed remarkable similarity in the smooth muscle configuration: ureteral muscle was clearly present, embedded in the bladder wall, corresponding to the unlabeled portion of the trigone in the Sm22CreR26RlacZ mouse (Fig. 5D-F). Hence, ureteral fibers make a contribution to the trigone,which is formed mainly from bladder muscle.

The trigone is formed predominantly from bladder muscle. (A)A sagittal section through a Sm22-Cre-R26RlacZ embryo at E14. lacZ-expressing mesenchymal cells are visible in the bladder, urethra and trigone (white arrow), but not in the ureter or Wolffian duct. (B)Section through the bladder and urethra of an adult Sm22-Cre-R26RlacZmouse showing descendents of the urogenital sinus mesenchyme that have differentiated in the bladder and urethra muscle. (C) Section through an adult Sm22-Cre-R26RlacZ mouse showing the ureter, which has few if any lacZ-expressing cells, and its path through the bladder muscle that is extensively labeled by the Sm22-Cre transgene. (D) A section through the intramural portion of the ureter in an Sm22-Cre-R26RlacZ adult. (E) A section from the same sample as in D, stained for smooth muscle alpha actin to reveal muscle of the intramural ureter, unlabeled by the Sm22-Cre transgene. (F) Section through a comparable level of a human embryo showing the path of the intramural ureter through the bladder muscle of the trigone. Magnification:×100 in A-C ×200 in D-F.

The trigone is formed predominantly from bladder muscle. (A)A sagittal section through a Sm22-Cre-R26RlacZ embryo at E14. lacZ-expressing mesenchymal cells are visible in the bladder, urethra and trigone (white arrow), but not in the ureter or Wolffian duct. (B)Section through the bladder and urethra of an adult Sm22-Cre-R26RlacZmouse showing descendents of the urogenital sinus mesenchyme that have differentiated in the bladder and urethra muscle. (C) Section through an adult Sm22-Cre-R26RlacZ mouse showing the ureter, which has few if any lacZ-expressing cells, and its path through the bladder muscle that is extensively labeled by the Sm22-Cre transgene. (D) A section through the intramural portion of the ureter in an Sm22-Cre-R26RlacZ adult. (E) A section from the same sample as in D, stained for smooth muscle alpha actin to reveal muscle of the intramural ureter, unlabeled by the Sm22-Cre transgene. (F) Section through a comparable level of a human embryo showing the path of the intramural ureter through the bladder muscle of the trigone. Magnification:×100 in A-C ×200 in D-F.

The structure of the trigone is likely to depend on intercalation of ureteral and bladder muscle. (A) A sagittal section through an E17 Pax2 +/+ embryo showing the point at which the ureteral longitudinal fibers join the bladder detrusor (yellow arrows). (B) A sagittal section through a Pax2 -/- littermate of that shown in A, showing the structure of the trigone region in the absence of the ureter. Note the abundant bladder and urethral muscle, and the tunnel through the bladder (red arrow) present in both wild type (A) and mutant (B). det,detrusor. Magnification: ×100.

The structure of the trigone is likely to depend on intercalation of ureteral and bladder muscle. (A) A sagittal section through an E17 Pax2 +/+ embryo showing the point at which the ureteral longitudinal fibers join the bladder detrusor (yellow arrows). (B) A sagittal section through a Pax2 -/- littermate of that shown in A, showing the structure of the trigone region in the absence of the ureter. Note the abundant bladder and urethral muscle, and the tunnel through the bladder (red arrow) present in both wild type (A) and mutant (B). det,detrusor. Magnification: ×100.

Ureters enter the trigone through a tunnel and ureteral fibers intercalate with bladder muscle

One piece of evidence supporting the idea that ureteral muscle is important for formation of the trigone is the observation that ureter agenesis results in an abnormally shaped ipsolateral hemitrigone. Ureteral muscle is thought to contribute extensively to the trigone itself and, according to the literature,the ureteral passageway to the trigone is encased in a sheath that is formed from ureteral musculature (Waldeyer,1892) (reviewed by Hutch,1972). Analysis of muscle differentiation in sagittal sections of wild-type E18 embryos revealed that the ureter passes through a tunnel in the bladder wall in parallel with blood vessels. Ureteral muscle fibers terminate in the trigone and intersect with ureteral and bladder muscle exclusively at its lateral edges. These findings suggest that the trigonal structure might be formed from this pathway of the ureter through the bladder and intercalation of the ureteral and urogenital sinus-derived fibers.(Fig. 6A). To further address this question, we analyzed trigone formation in the absence of the ureter in Pax2 mutants, which display apparently normal urogenital sinus differentiation but lack ureters and kidneys owing to agenesis of the caudal Wolffian duct. The trigone in the Pax2 mutant shown(Fig. 6B) contains bladder muscle that appeared to completely encircle the bladder neck. Interestingly,both in Pax2 mutants and in wild-type littermates, a gap was present in the bladder wall, which probably corresponds to the ureteral tunnel. In wild-type mice, the tunnel contained the intramural ureter and blood vessels that pass through the muscle and submucosa into the urothelium. In Pax2 mutants, the tunnel was also present, but contained only blood vessels owing to the absence of the ureter. The presence of the ureteral tunnel in the absence of ureters indicates that it is almost certainly derived from the bladder/trigone. The observation that intercalation of ureteral and bladder muscle occurs only at the lateral sides of the trigone is consistent with the requirement for the ureter to maintain the raised triangular structure normally associated with the trigone, explaining why the absence of the ipsolateral ureter results in deformation of the trigone.

Models of trigone formation. (A) Old model of trigone formation, showing the trigone to be continuous with the ureters (green),formed in large part from ureteral fibers that fan out across the surface generating the inter-ureteric ridge and Bell's muscle. Note that the trigone has been considered to form independently of the bladder. (B) Current model of trigone formation, showing a small contribution from ureteral fibers(green) and the bulk of the structure derived from bladder muscle and the space around the ureter that functions as a tunnel.

Models of trigone formation. (A) Old model of trigone formation, showing the trigone to be continuous with the ureters (green),formed in large part from ureteral fibers that fan out across the surface generating the inter-ureteric ridge and Bell's muscle. Note that the trigone has been considered to form independently of the bladder. (B) Current model of trigone formation, showing a small contribution from ureteral fibers(green) and the bulk of the structure derived from bladder muscle and the space around the ureter that functions as a tunnel.


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5. The scale of the burden of urinary incontinence

5.1. Overview

Data from 2006� reveal that 𢏁.3 million people in England sought help for incontinence problems. The number had risen to 2.3 million in 2010�. Urinary incontinence increases with age from 14% in individuals aged 65� years to 45% in those aged 85 years or over [64]. The care of older and disabled people in an ageing population presents a major challenge: the management of bladder (and bowel) function is fundamental to the standard of care that they receive. It is difficult or impossible for those affected to maintain a reasonable quality-of-life and urinary incontinence is a major reason for sufferers to seek residential care [65].

In a study of 430 new admissions to nursing homes in the US, 39% of patients aged 65 years or over suffered from daytime urinary incontinence [66]. In this setting, catheterization is only recommended as a last resort, because of its high incidence of urinary tract infections. Elderly patients are managed by using incontinence pads, but immobile patients lying on wet pads develop pressure sores. Choosing the lesser of two evils, the development of a pressure sore is accepted as an indication for catheterization.

In England, Wales and Northern Ireland (and there is no reason to suppose that the situation is significantly different in Scotland), indwelling Foley catheters are used by 3% of people living in the community and 13% of care home residents [67].

5.2. Incidence of adverse events

It is a profoundly disturbing statistic that healthcare-associated urinary tract infections are estimated to have caused 13 088 deaths in hospitals in the US in 2002 [68]. Assuming that 80% of these were due to catheter-induced infections [16], that equates to 10 470 deaths. The population of the US is 4.98-times that of the UK, so the corresponding annual number of deaths in the UK is probably at least 2100.

In a postal survey to determine the incidence and morbidity of long-term catheterization in a typical National Health Service setting [69], there were 506 referrals from a cohort of 457 patients over a 6-months period. From these referrals, 54 patients were selected for detailed study: 48% experienced catheter blockage, 37% reported urine by-passing the catheter and 30% noted haematuria.

Catheter-associated urinary tract infections not only place heavy demands on healthcare resources, but their treatment also raises profound concern regarding the development of antimicrobial resistance and they cause immense distress and social problems for the sufferers, their carers and their communities.

5.3. Economic implications

A rigorous analysis of economic costs of urinary retention, incontinence and catheterization is beyond the scope of this review. It may be helpful, however, to give some insight into the orders of magnitude involved, as follows:

In 2008, the world market for urinary continence care devices of all kinds (mainly catheters and pads) was estimated to be US$1.8 billion per year, growing at 𢏇% per year of this, Foley catheters accounted for ∼US$380 million in 2007 [70]. The costs of the relevant devices are, however, small in relation to those of the clinical and societal consequences of incontinence and retention and its management by long-term catheterization. In the US in 2002, urinary tract infections were responsible for over 7 million physician visits they accounted for more than 100� hospital admissions, mostly for pyelonephritis and the direct and indirect costs associated with community-acquired urinary tract infections exceeded an estimated US$1.6 billion [71]. In 1997, �% of all community-prescribed antibiotics in Germany were dispensed for urinary tract infections, at an estimated cost of over US$1 billion [72]. In 1991, it was estimated that an episode of nosocomial bacteriuria added US$500� to the direct cost of acute-care hospitalization [73].

The use of incontinence pads could be greatly reduced if there were a satisfactory alternative to the Foley catheter. In addition to the fact that the use of pads is difficult or impossible to conceal, thus tending to make sufferers socially reclusive, there is the further problem of progressive deterioration of the condition of their skin. Unless pads are changed so frequently that the skin is kept dry, it tends to become macerated, leading to pressure sores and, with repeated drenching, the problem becomes chronic and unmanageable. The cost of pads to the NHS in England increased from ꍷ million in 2006/2007 to 򣄡 million in 2010/2011, so that their use has begun to be restricted. Until 2013, the allocation was based on clinical need. Now it is often based on financial considerations and, as a result, the allowance per patient can be as few as four disposable continence products in 24 h [64]. This rationing perversely ignores the downstream costs of treating the resulting increase in the incidence of pressure ulcers, many of which are due to the use of pads and which, in the UK, was already between ਱.4–ਲ.1 billion annually in 2004, accounting for 4% of total NHS expenditure [74].

Clearly it is impossible to make a simple and accurate calculation of the financial cost attributable to catheter-associated urinary tract infections. According to hospital episode statistics, there were 281� finished consultant episodes of serious adverse events related to urinary tract infections in National Health Service hospitals (and private hospitals undertaking work for the NHS) in England and Wales in 2012� [75]. It is reasonable to assume that 80% of these episodes (225,036) were due to catheter-induced infections [16]. Data for 2001 show that infected patients, on average, incurred UK hospital costs 2.9-times higher than uninfected patients, at that time equal to an additional � per patient [76]. Between 2001�, the UK consumer price index increased by a factor of 1.48 [77]. Admittedly this may not be a very good index of healthcare cost inflation, but no better measure seems to be available. Thus, the additional UK National Health Service cost of each catheter-associated urinary tract infection must be in the order of � so, by this calculation, the total annual cost of all episodes must be at least ਱.0 billion.

Data for Scotland in 1999 [78] gave an estimated 򣄥 million as the additional annual cost of treating catheter-associated urinary tract infections. Scotland accounts for 𢏈% of the UK population and financial inflation from 1999 to 2015 was �%. This calculation indicates that the total current UK cost of catheter-associated urinary tract infections is ∼ਲ.5 billion per year.

Thus, it can be concluded that the annual total additional UK cost of catheter-associated urinary tract infections probably now lies somewhere between ਱.0–ਲ.5 billion.

For the US, it was recently estimated that healthcare-acquired infections account for nearly US$45 billion per year in direct hospital costs [79]. Assuming that 80% were due to catheter-induced infections [16], the total annual cost must be ∼US$36 billion.

Prior to 1 October 2008, hospitals in the US were able to recover additional payment to compensate for the extra cost of treatment of catheter-associated urinary tract infections. Then the Medicare rule was changed and reimbursement for this was stopped [80]. Whether or not this was justified, the rationale was that catheter-associated urinary tract infection could reasonably be prevented through the application of evidence-based guidelines. Whatever the other effects of this change in the rule have been, however, it is perhaps surprising that it does not seem yet to have been seen as an incentive to develop a better catheter.

Of course, the availability of a better catheter would not completely eliminate catheter-associated urinary tract infections but, even if only 50% of its potential could be achieved, the annual savings would be in the order of 򣔀 million in the UK and US$18 billion in the US. Furthermore, over 1000 deaths would probably be avoided each year in the UK and over 5000 in the US.


Urinary tract infection: How bacteria nestle in

Almost every second woman suffers from a bladder infection at some point in her life. Also men are affected by cystitis, though less frequently. In eighty percent of the cases, it is caused by the intestinal bacterium E. coli. It travels along the urethra to the bladder where it triggers painful infections. In Nature Communications researchers from the University of Basel and the ETH Zurich explain how this bacterium attaches to the surface of the urinary tract via a protein with a sophisticated locking technique, which prevents it from being flushed out by the urine flow.

Many women have already experienced how painful a bladder infection can be: a burning pain during urination and a constant urge to urinate are the typical symptoms. The main cause of recurrent urinary tract infections is a bacterium found in the normal flora of the intestine, Escherichia coli. The bacteria enter the urinary tract, attach to the surface and cause inflammation.

The teams of Prof. Timm Maier at the Biozentrum and Prof. Beat Ernst at the Pharmazentrum of the University of Basel, along with Prof. Rudolf Glockshuber from the Institute of Molecular Biology and Biophysics at the ETH Zurich, have now discovered how bacteria adhere to the urinary tract under urine flow via the protein FimH and subsequently travel up the urethra.

Intestinal bacterium adheres to the cell surfaces with the protein FimH

The pathogen has long, hairlike appendages with the protein FimH at its tip, forming a tiny hook. This protein, which adheres to sugar structures on the cell surface, has a special property: It binds more tightly to the cell surface of the urinary tract the more it is pulled. As strong tensile forces develop during urination, FimH can protect the bacterium from being flushed out.

"Through the combination of several biophysical and biochemical methods, we have been able to elucidate the binding behavior of FimH in more detail than ever before," says Glockshuber. In their study, the scientists have demonstrated how mechanical forces control the binding strength of FimH. "The protein FimH is composed of two parts, of which the second non-sugar binding part regulates how tightly the first part binds to the sugar molecule," explains Maier. "When the force of the urine stream pulls apart the two protein domains, the sugar binding site snaps shut. However, when the tensile force subsides, the binding pocket reopens. Now the bacteria can detach and swim upstream the urethra."

Drugs against FimH to combat urinary tract infections

Urinary tract infections are the second most common reason for prescribing antibiotics. Yet, in times of increasing antibiotic resistance, the focus moves increasingly to finding alternative forms of treatment. For the prevention and therapy of E. coli infections, drugs that could prevent the initial FimH attachment of the bacteria to the urinary tract could prove to be a suitable alternative, as this would make the use of antibiotics often unnecessary.

This opens up the possibility of reducing the use of antibiotics and thus preventing the further development of resistance. Prof. Ernst, from the Pharmazentrum of the University of Basel, has been working intensively on the development of FimH antagonists for many years. The elucidation of the FimH mechanism supports these efforts and will greatly contribute to the identification of a suitable drug.


The Role(s) of Cytokines/Chemokines in Urinary Bladder Inflammation and Dysfunction

Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain syndrome characterized by pain, pressure, or discomfort perceived to be bladder related and with at least one urinary symptom. It was recently concluded that 3.3–7.9 million women (>18 years old) in the United States exhibit BPS/IC symptoms. The impact of BPS/IC on quality of life is enormous and the economic burden is significant. Although the etiology and pathogenesis of BPS/IC are unknown, numerous theories including infection, inflammation, autoimmune disorder, toxic urinary agents, urothelial dysfunction, and neurogenic causes have been proposed. Altered visceral sensations from the urinary bladder (i.e., pain at low or moderate bladder filling) that accompany BPS/IC may be mediated by many factors including changes in the properties of peripheral bladder afferent pathways such that bladder afferent neurons respond in an exaggerated manner to normally innocuous stimuli (allodynia). The goals for this review are to describe chemokine/receptor (CXCL12/CXCR4 CCL2/CCR2) signaling and cytokine/receptor (transforming growth factor (TGF-β)/TGF-β type 1 receptor) signaling that may be valuable LUT targets for pharmacologic therapy to improve urinary bladder function and reduce somatic sensitivity associated with urinary bladder inflammation.

1. Lower Urinary Tract (LUT)

1.1. Anatomy

The LUT (bladder and urethra) is a division of the renal system that functions to passively store kidney byproducts until it is appropriate to void. To accomplish this, the urinary bladder is a muscular and membranous organ whose structure embodies its reservoir function. Its external features can be organized into an apex, fundus, body, and neck. The apex, or vertex, is on the anterior surface of the urinary bladder and is associated with ligament remnants attached to the umbilicus [1]. The posterior surface is the fundus and its most inferior aspect is termed the base of the urinary bladder [1]. The body typically represents the area between the apex and the fundus and the bladder neck is the most caudal aspect of the inferior bladder surface that is perforated by the internal urethral orifice [1].

The urinary bladder wall is composed of three layers: tunica mucosa, tunica muscularis propria, and tunica serosa/adventitia. The tunica mucosa consists of transitional epithelium and a lamina propria. Transitional epithelial cells in the urinary bladder are termed the urothelium and are arranged in basal, intermediate, and apical cell layers. Basal cells are monolayers directly attached to the basement membrane [2]. Intermediate cells are generally larger in diameter than basal cells and range from one to multiple cell layers depending on the species [2]. The apical, or umbrella, cells are hexagonal in shape and range from 25 to 250 μm depending on urinary bladder distention [2, 3].

Several distinct features of the luminal surface of umbrella cells establish antiadherence and an impermeable barrier characteristic of the urinary bladder mucosa. First, tight junction complexes comprised of occludin and claudin proteins regulate paracellular transport between adjacent umbrella cells [3]. The apical membrane is also occupied by uroplakin, a crystalline plaque cell surface protein that forms an asymmetric unit membrane to maintain impermeability during bladder expansion [4]. Lastly, a layer of proteoglycans on the mucosal surface of umbrella cells serves as an antiadherence factor and provides yet another physical barrier between urinary constituents and the lamina propria [5].

The extracellular matrix of the lamina propria is deep to the basement membrane of the urothelium and contains a diverse array of interstitial cells, nerve terminals, and vasculature [3, 6]. It has been suggested that the lamina propria may have an important role in integrating epithelial and smooth muscle function due to its innervations and proximity to the urothelium and tunica muscularis propria [6]. The tunica muscularis propria consists of three smooth muscle layers termed the detrusor. The internal and external layers are arranged longitudinally, whereas those in the middle are circular [7, 8]. The smooth muscle cells in the muscularis propria retain their classic spindle shape and are bundled together by collagen-rich connective tissue [7]. External to the muscularis propria, the tunica serosa surrounds the superior and lateral surfaces of the urinary bladder wall, whereas the retroperitoneal aspects contain a vascular, loose connective tissue termed the tunica adventitia [8].

Caudal to the inferior surface of the urinary bladder is the urethra. Similar to the urinary bladder wall, the urethral wall is composed of a tunica mucosa, tunica muscularis propria, and tunica adventitia. The tunica mucosa consists of transitional epithelium proximal to the urinary bladder followed by nonkeratinized, stratified squamous epithelium distally [8, 9]. The tunica muscularis propria is composed of inner and outer smooth muscle arranged longitudinally and circularly, respectively [8]. In the male urethra, the circular smooth muscle fascicles join with urinary bladder smooth muscle at the urethrovesical junction to form the internal urethral sphincter [8, 9]. The smooth muscle fascicles along the proximal female urethra, however, do not appear to anatomically arrange into a sphincter [9]. Skeletal muscle of the urethral wall forms the external urethral sphincter and extends along the membranous urethra in males to generate voluntary pressure during bladder filling [10]. The skeletal muscle fibers in the female urethra join to form an “external” urethral sphincter comprised of a sphincter urethrae, compressor urethrae and sphincter urethrovaginalis to provide urinary continence through urethral and vaginal closure [11].

1.2. Neural Control

The LUT is regulated by supraspinal, spinal, and peripheral nervous system (PNS) input to maintain “switch-like” patterns of storage and elimination activity and has been previously reviewed in greater detail [10]. Briefly, bladder wall mechanoreceptors initiate visceral afferent (Aδ fibers) activity during the storage phase that synapse on spinal interneurons [10, 12]. Spinal reflex pathways then facilitate storage by directly enhancing thoracolumbar sympathetic outflow and somatomotor discharge or ascending, in some species, to keep metencephalic integration centers [10, 12].

Spinal interneurons activate preganglionic sympathetic fibers from the intermediolateral cell column of the lower thoracic (T10) through upper lumbar (L2) spinal cord that form thoracic and lumbar splanchnic nerves [13, 14]. The preganglionic fibers then synapse on the prevertebral inferior mesenteric ganglia or paravertebral ganglia and travel along the hypogastric and pelvic nerves, respectively [10]. Adrenergic neurotransmission on the urinary bladder smooth muscle β-adrenergic receptors promotes bladder wall relaxation and accommodation [13]. Bladder filling is also facilitated by the activation of

-adrenergic receptors on the internal urethral sphincter resulting in contraction of the urethral outlet [13]. Spinal reflex pathways not only enhance sympathetic outflow but also -motoneuron discharge from Onuf’s nucleus in the ventrolateral horn of the sacral (S2–S4) spinal cord [12]. Propagation of this signal along the pudendal nerve to the external urethral sphincter elicits skeletal muscle contraction by activating nicotinic acetylcholine receptors to provide voluntary control over urinary continence [13].

Upon reaching the tension threshold, bladder afferents (Aδ fibers) bypass local spinal reflexes and ascend to the mesencephalic periaqueductal gray (PAG). Unlike the reflexes underlying the storage phase, the elimination phase relies on supraspinal circuitry as evidenced by voiding dysfunction following lower thoracic spinal cord injury [14, 15]. After cortical processing, the PAG sends excitatory input to a region in the dorsolateral pontine tegmentum termed the pontine micturition center (PMC) [16]. The PMC then sends descending cortical projections that synapse on preganglionic parasympathetic neurons and inhibitory interneurons in the sacral spinal cord [14, 16].

The preganglionic parasympathetic fibers arise from the intermediolateral cell column of the sacral (S2–S4) spinal cord to form pelvic splanchnic nerves. Upon coursing through and exiting the hypogastric and pelvic plexus, the fibers join the pelvic and pudendal nerves to synapse on terminal ganglia and innervate the detrusor smooth muscle and urethra [12, 13]. Cholinergic and nonadrenergic/noncholinergic neurotransmission on the urinary bladder smooth muscle promotes bladder wall contraction by activating muscarinic acetylcholine receptors and purinergic receptors, respectively [14]. Elimination of urine is also facilitated by nitric oxide release onto the internal urethral sphincter resulting in a relaxation of the urethral outlet [14]. The PMC not only augments parasympathetic outflow but also attenuates preganglionic sympathetic and -motoneuron discharge to the LUT [16]. The descending cortical projections terminating on inhibitory interneurons in the sacral spinal cord prevent excitatory input into the urethral sphincters resulting in dilation of the urethral orifice and continuous flow of urine. As distention of the urinary bladder decreases during the elimination phase, ascending excitation to the dorsolateral metencephalon is diminished and the storage phase is once again switched on.

1.3. Symptoms and Dysfunction

The terminology used in the following section is consistent with the standardization report of LUT symptoms and function by the International Continence Society and will refer to their definitions when appropriate [17]. Similar to other clinical indications, LUT symptoms are the patient’s qualitative representation of a purported condition. These symptoms, in particular, refer to a spectrum of LUT functions that include storage, elimination, and postmicturition disturbances.

Symptoms associated with the storage phase include, but are not limited to, “increased frequency, urgency, and incontinence” [17]. The complaint of increased urinary frequency is prevalent among both men and women with LUT dysfunction and has been suggested to affect an individual’s quality of life as demonstrated by a strong correlation between frequency and bothersome endorsements [18]. Increased urgency is a complaint of the “sudden compelling desire to pass urine” that may be accompanied by pain, pressure, or discomfort associated with the LUT [17]. Lastly, urinary incontinence includes a complaint of the “involuntary leakage of urine” and may manifest in various forms and severities [17]. It is important to note that incontinence is not representative of one particular LUT dysfunction but rather can arise from multiple sources including stress, comorbid disorders, and congenital abnormalities [19].

Symptoms associated with the elimination phase include “hesitancy, slow or intermittent stream, straining, and terminal dribble” [17]. These symptoms generally involve complaints of the initiation and continuation of voiding and alterations to their urine stream and appear to be more prevalent in men compared to women [17, 18]. Symptoms associated with the postmicturition phase occur after voiding and include “incomplete emptying and postmicturition dribble” [17]. Although equally bothersome, postmicturition dribble may be more prevalent in men, whereas, in women, incomplete emptying may be more prevalent [18]. As briefly mentioned above, LUT symptoms are not confined to urodynamic disturbances but may also include unpleasant sensations of pain or discomfort during storage or elimination. These sensations are generally perceived to emanate from the urogenital organs and may exacerbate storage and elimination symptoms [20].

2. Bladder Pain Syndrome (BPS)/Interstitial Cystitis (IC)

2.1. Background

LUT signs and symptoms resembling what is currently termed BPS/IC have been documented throughout history and its perspective has been previously reviewed in detail [21]. Briefly, Drs. Philip Syng Physick and Joseph Parish first recognized an inflammatory condition called tic douloureux of the bladder whose symptoms included chronic urinary frequency, urgency, and pelvic pain [22]. Skene [23] expanded the cystoscopic features of this concept in the late 19th century and introduced the term IC which included ulceration of the mucous membrane and inflammation within the bladder wall. Focal, ulcerative bleeding in the urinary bladder wall remained a hallmark of IC due, in part, to the work of Hunner [24] in the early 20th century [21]. Many patients, however, were misdiagnosed as current estimates suggest only 5–7% of those with BPS/IC present with bladder ulcerations [21, 25].

In the absence of a formal classification for IC, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) attempted to standardize its research definition in 1987 by establishing a diagnostic criteria [26]. The criteria included the presence of “glomerulations on cystoscopic examination or a classic Hunner ulcer, pain associated with the bladder, urinary urgency” and eighteen exclusion conditions [21, 26]. After several iterations and international consultations, the term IC was expanded to include BPS [27, 28]. The patient selection for BPS is based on “chronic pelvic pain, pressure, or discomfort perceived to be related to the urinary bladder accompanied by at least one other urinary symptom such as persistent urge to void or frequency,” whereas IC is “reserved for cystoscopic and histological features” [17, 28]. At this time, the terms BPS and BPS/IC are analogous and are defined by the American Urological Association Interstitial Cystitis Guidelines Panel as at least six weeks of LUT symptoms and unpleasant sensations perceived to be related to the urinary bladder and with no other clinically identifiable sources [27].

2.2. Epidemiology

The epidemiology of BPS/IC is limited due to the absence of standardized definitions, markers, and examinations [21, 25, 29, 30]. Taking into account this variability, it is estimated that there is a 5 : 1 female-to-male ratio among BPS/IC patients [21, 25]. It is estimated that 300 per 100,000 women worldwide suffer from BPS/IC [21]. In the United States alone, 3.3 to 7.9 million women are estimated to meet the criteria for BPS/IC [30]. As expected, BPS/IC puts an enormous financial burden on the individual and economy as a whole. Health care costs for an individual with BPS/IC range from 4 to 7 thousand dollars per year, while the economic burden approaches 500 million dollars per year in lost productivity and therapeutics [21, 31].

2.3. Pathophysiology

While the primary insult underlying BPS/IC is not known, it has been suggested that the pathophysiology is a “vicious circle” involving uroepithelial dysfunction, inflammation, afferent nerve hyperexcitability, and visceral hyperalgesia and allodynia (Figure 1) [32]. This section will explore the said mechanisms that have been proposed to feedforward to promote the chronicity of LUT symptoms observed in BPS/IC [32].


Potential etiologic cascade and pathogenesis underlying painful bladder syndrome (BPS)/interstitial cystitis (IC). It is likely that BPS/IC has a multifactorial etiology that may act predominantly through one or more pathways resulting in the typical symptom-complex. There is a lack of consensus regarding the etiology or pathogenesis of BPS/IC but a number of proposals include a “leaky epithelium,” release of neuroactive compounds at the level of the urinary bladder with mast cell activation, “awakening” of C-fiber bladder afferents, and upregulation of inflammatory mediators including cytokines and chemotactic cytokines (chemokines). Inflammatory mediators can affect CNS and PNS neural circuitry including central “wind-up” and nociceptor sensitization resulting in chronic bladder pain and voiding dysfunction. BPS/IC is associated with diseases affecting other viscera and pelvic floors. See text for additional details. Figure adapted from [32].

The urothelium is a specialized, stratified epithelium that when intact provides a nonadherent, passive barrier through tight junction proteins, plaque proteins, and surface proteoglycans [2]. Any perturbation to the components of this permeability barrier may lead to increased infiltration into the bladder wall and exposure of the interstitium to urinary constituents [33–36]. The diffusion of urinary constituents like potassium into the bladder interstitium may depolarize muscle and nerve cells, inflame tissues, degranulate mast cells, and cascade to the development of LUT symptoms (Figure 1) [35]. Uroepithelial dysfunction specific to BPS/IC, however, remains controversial. For example, Chelsky et al. [37] demonstrated that the permeability in IC was comparable to the variation seen in symptom-free controls, whereas Parsons et al. [36] demonstrated abnormal permeability and potassium absorption in those with IC [21]. The abundance of studies for or against uroepithelial dysfunction in BPS/IC suggests that it may not be a primary insult but rather may occur in a subset of patients to exacerbate LUT symptoms [21].

In addition to the uroepithelial disruption, visceral inflammation also remains a central pathological process in BPS/IC and has been suggested to underlie the development of LUT symptoms (Figure 1). Inflammation within the urinary bladder viscera is characterized by increased vasculature, mucosal irritation that may result in barrier dysfunction, and infiltration of inflammatory mediators [38, 39]. The proliferation and activation of mast cells, in particular, have received considerable attention in the urinary bladder immune response [32]. Mast cells secrete vasoactive chemicals to promote innate and autoimmunity and their increased activity has been widely demonstrated in BPS/IC [40–43]. The subsequent exposure in the bladder interstitium to vasoactive chemicals, inflammatory mediators, and neuropeptides from visceral inflammation may lead to afferent nerve hyperexcitability and neurogenic inflammation (Figure 1) [44–46].

The loss of inhibition on peripheral afferents (Aδ and C fibers) increases input into the spinal cord and may eventually promote central sensitization [32]. An unregulated state of central and peripheral reactivity causes “wind-up” which is observed clinically as hyperalgesia and allodynia (Figure 1). In BPS/IC, hyperalgesia and allodynia are characterized by an elevated state of urinary bladder sensation that may cause pain, pressure, or discomfort and may result in increased urinary frequency and urgency [38]. The “vicious circle” continues as mast cell degranulation and infiltration of mediators from uroepithelial dysfunction and/or visceral inflammation sustain peripheral and central sensitization to establish visceral hyperalgesia/allodynia and chronic LUT symptoms (Figure 1) [32].

2.4. Animal Models

Numerous animal models have been implemented to determine the onset and chronicity of LUT dysfunctions like BPS/IC. While one model cannot currently account for the constellation of symptoms in BPS/IC, they each aid in identifying distinct mechanisms underlying part of its pathophysiology. This section will explore a naturally occurring cystitis model in felines and focus its review on experimental models of cystitis induced chemically. It is important to note that models of BPS/IC are not limited to what will be discussed in this section and exhaustive reviews have been previously published [47–49].

The natural development of spontaneous LUT symptoms has been documented in cats for several decades and is termed feline interstitial cystitis (FIC) [47, 50]. Though the primary insult for FIC is not known, the pathophysiology has marked similarities to BPS/IC including uroepithelial dysfunction and visceral inflammation. Cats with FIC have been shown to have a disruption to the epithelial cytoarchitecture that increased diffusion and infiltration of urinary constituents [51, 52]. Uroepithelial dysfunction in FIC further led to a peripheral upregulation of neuropeptides and inflammatory mediators that altered bladder afferent soma size and increased input to the central nervous system (CNS) [53]. As previously discussed, the alterations to central and peripheral reactivity following uroepithelial dysfunction and/or visceral inflammation may promote the development of LUT symptoms that is observed in FIC and, by extension, BPS/IC [32, 38, 53].

Despite these pathophysiological similarities, FIC as a model for BPS/IC is limited due to its spontaneity and epidemiology. Investigators are practically and financially restricted to structural and functional alterations following its spontaneous induction and thus inadequately define insults preceding the development of FIC [49]. Furthermore, unlike BPS/IC, FIC occurs irrespective of biological sex [49]. While this may be due to a misdiagnosis of BPS/IC in males, one cannot discount hormonal differences that may affect LUT symptoms in humans [54, 55].

LUT symptoms have also been induced by an assortment of chemical irritants including, but not limited to, hydrochloric acid, acetic acid, protamine sulfate (PS), and cyclophosphamide (CYP). The inflammation induced by intravesical instillation of irritants like hydrochloric acid and acetic acid helps reveal the anatomical, organizational, and functional alterations attributable to the visceral immune response [47]. Specifically, the functional and histological features following acid instillation are similar to a BPS/IC subset and include urothelial hyperplasia, bladder ulceration, mucosal edema, inflammatory cell infiltration, and the development of LUT symptoms [56, 57]. Though acid instillation allows for a more controlled environment than FIC, the studies must be interpreted cautiously as the degree of inflammation resulting from exogenous irritants may not be representative of the naturally occurring BPS/IC [47].

Unlike acid instillation, PS lacks a pervasive inflammatory element but rather disrupts uroepithelial barrier function by targeting bladder surface proteoglycans [58]. Similar to the uroepithelial dysfunction observed in FIC, PS instillation is sufficient to induce LUT symptoms [59]. More recently, PS has been used in conjunction with bacterial induced cystitis. Instillation of both PS and E. coli lipopolysaccharide to, respectively, damage the urothelium and induce a visceral inflammatory cascade may help clarify the interaction(s) of multiple processes underlying LUT symptoms in BPS/IC [47, 60].

CYP is an antineoplastic prodrug that requires enzymatic activation to release phosphoramide mustard and the byproduct acrolein [61, 62]. A known adverse toxicity following systemic CYP administration is hemorrhagic cystitis [62]. Hemorrhagic cystitis is considered to arise from the bladder mucosal walls contact with acrolein, which has been shown to increase vascular permeability and result in bladder ulceration and hypertrophy [63]. In addition to hemorrhagic cystitis, systemic CYP treatment causes functional and histological changes similar to BPS/IC including mucosal edema, uroepithelial dysfunction, inflammatory cell infiltration, afferent nerve hyperexcitability, and the development of LUT symptoms [45, 64–67]. CYP administration also produces behavioral alterations consistent with the development of viscerosomatic pain including decreased breathing rate, closing of the eyes, and rounded back postures [66]. While the urinary bladder inflammatory response following systemic CYP administration is greater than what is observed in BPS/IC, this experimental model of cystitis is appealing because of its route of administration (intraperitoneal) and the chronicity and reproducibility of histopathological and functional alterations [47].

3. Inflammatory Mediators in Urinary Bladder Inflammation

We have hypothesized that pain associated with BPS/IC involves an alteration of visceral sensation/bladder sensory physiology. Altered visceral sensations from the urinary bladder (i.e., pain at low or moderate bladder filling) that accompany BPS/IC [68–72] may be mediated by many factors including changes in the properties of peripheral bladder afferent pathways such that bladder afferent neurons respond in an exaggerated manner to normally innocuous stimuli (allodynia). These changes may be mediated, in part, by inflammatory changes in the urinary bladder (Figure 1). Among potential mediators of inflammation, neurotrophins (e.g., nerve growth factor, NGF) have been implicated in the peripheral sensitization of nociceptors [73–75]. Proinflammatory cytokines also cause sensitization of polymodal C-fibers [74] and facilitate A-beta input to the spinal cord [76, 77]. Several studies from our laboratory have demonstrated increased expression of cytokines and chemokines (chemotactic cytokines) and the beneficial effects of receptor blockade in the urinary bladder after CYP-induced bladder inflammation [64]. In the next sections, we will present a summary of recent studies from our laboratory that addresses the role(s) of two chemokine/receptor pairs (CXCL12/CXCR4 CCL2/CCR2) and the cytokine/receptor pair (transforming growth factor (TGF-β)/TGF-β type 1 receptor) in urinary bladder inflammation and somatic sensitivity in a CYP rat model of urinary bladder inflammation.

Using the CYP-induced bladder inflammation model, we aimed to characterize further the role of inflammatory chemicals in the development and/or maintenance of neuronal sensitization and chronic pain states associated with BPS/IC. Inflammatory chemicals are released at sites of injury and inflammation by resident and infiltrating immune cells and endothelial and parenchymal cells. Proinflammatory molecules act to heal the injured/inflamed area and also to sensitize nociceptive neurons, thus increasing the pain response in order to prevent further insult [78]. While initial immune activation and sensitization of sensory neurons is protective, prolonged inflammatory processes and sensory sensitization occurring after tissue healing are associated with chronic pain syndromes, including BPS/IC. Various cytokines and chemokines have been detected in the urine and urinary bladder in models of cystitis and patients with BPS/IC and therefore may represent novel therapeutic targets or biomarkers for the syndrome.

4. Chemokines

4.1. Background

Chemokines are a large family of structurally and functionally related proteins that are important mediators of immune responses, inflammatory processes, and nociception. In the immune response, chemokines facilitate tissue recovery by causing the extravasation of leukocytes from blood plasma to the site of injury. Chemokine receptors present on leukocytes sense increasing chemotactic concentration gradients and facilitate cellular motility towards them [79].

Chemokines are small, secreted proteins of approximately 100 amino acids in length that comprise 4 subfamilies: CC, CXC, CX3C and C (review see [80]). Each subfamily is named for the first cysteine residue motif from its amino terminus. Families of chemokines assert their actions by signaling via related G-coupled protein receptors. Within each subfamily, receptor and ligand pairing is not mutually exclusive in other words, multiple ligands can bind the same receptor and vice versa (for review see [80]). The complexity of chemokine receptor binding presents challenges when examining the functional role of chemokine signaling. Despite difficulties, determining the role of chemokines and their receptors in both control and pathological states could provide insights to possible therapeutic interventions in a variety of chronic pain conditions including BPS/IC.

4.2. Chemokines and Peripheral Sensitization

Inflammatory mediators such as proinflammatory cytokines (e.g., tumor necrosis- (TNF-) , interleukin- (IL-)6, IL-1β), COX-2, NGF, protons, prostaglandins, and bradykinin have been implicated in the direct sensitization of nociceptive afferents [81]. Traditionally, chemokines were not thought to assert direct effects on primary sensory neurons. Rather, chemokine/receptor interaction on the plasma membrane of leukocytes was thought to stimulate leukocyte release of nociceptive mediators via GCPR signaling mechanisms [78]. However, electrophysiological, expressional, and functional pain studies have demonstrated the possibility of direct chemokine-mediated neuronal hypersensitivity and pain. For example, in vitro, exogenous chemokine application can physiologically alter sensory neurons by changing membrane potentials [82], decreasing thresholds for action potential generation [82], increasing excitability, and evoking discharges [82, 83]. Various chemokines, including CXCL12, can modulate calcium ion currents in cultured DRG cells, potentially facilitating hyperexcitability [84–87]. In a neuronal injury model, chronic compression of the DRG elicits a depolarizing response to chemokines that was not detected in control (noncompressed) DRG [83]. Chemokine-mediated sensitization may involve members of the transient receptor potential family, including TRPV1. Various chemokines and receptors colocalize with neuronal TRPV1 as well as neuropeptides released in a TRPV1-dependent manner [87–90].

Following nerve injury or inflammation, expression of chemokines and associated receptors increases significantly in macrophages, infiltrating T cells, sensory neurons, and glia [79, 84, 85, 89, 91–93]. Additionally, increased neuronal activity, as would occur during injury or inflammation, has been shown to induce chemokine transcription in cultured DRG neurons [94]. Cytokines such as IL-1 can increase chemokine expression in neurons and astrocytes [95–97]. Chemokine expression in, and subsequent secretion from the various cell types (e.g., leukocytes, endothelial cells, neurons, or parenchymal cells) would enable chemokines, by diffusion, to interact with functional chemokine receptors on DRG neurons thus facilitating hyperexcitability changes such as those described above.

4.3. Chemokines and Central Sensitization

Increased primary afferent signaling can induce organizational and neurochemical changes in spinal cord synapses that underlie the phenomenon, central sensitization, which may contribute to chronic pain syndromes. During central sensitization, intensely heightened peripheral input decreases thresholds necessary to elicit action potentials in dorsal horn neurons. An increase in nociceptive neurotransmitter (e.g., SP and CGRP) release into the dorsal horn could increase the activity of spinal neurons that mediate both local reflexes and ascend to higher brain centers, thus facilitating the perception of pain [78, 87, 98].

Chemokine/receptor signaling may contribute to central sensitization via activation of either the peripheral or central afferent limbs of pain pathways [78, 98]. Chemokine activation of peripheral DRG neurons was described in the previous section. Additionally, chemokine application can evoke SP and CGRP release from DRG neurons that could cause chemokine-mediated central effects indirectly [87, 99]. Evidence suggests that chemokines may have direct central effects also. Jung et al. [89] detected large dense-core vesicles containing both CCL2 and CGRP in TRPV1-expressing DRG neurons. Dansereau et al. [88] demonstrated calcium-evoked release of CCL2 following incubation of DRG neurons with potassium or capsaicin. CCL2 may traffic anterogradely from the soma of peripheral sensory neurons and is increased in the supernatant following intense stimulation of mechanically injured DRG neurons [100]. Additionally, CCL2 application increases the frequency of spontaneous EPSCs in superficial dorsal horn neurons [101]. Chemokines released from primary afferent central terminals could exert either direct activation of superficial dorsal horn neurons via functional chemokine receptor expression or indirect sensitization via activation of microglia and astrocytes that subsequently release nociceptive mediators.

Chemokine receptors, including CCR2, and chemokines are detected in dorsal horn neurons and activated astrocytes and microglia in numerous models of neuropathic pain including peripheral or central nerve damage or tissue inflammation [102–107]. Chemokine cross signaling between bladder sensory afferents and microglia or astrocytes could modulate symptoms of BPS/IC especially considering that peripheral injury or inflammation (e.g., bladder) can induce central glial activation.

4.4. Chemokines and Nociception

Studies investigating nociceptive behavior illustrate a strong relationship between chemokines and pain. Exogenous administration of chemokines induces thermal hyperalgesia and mechanical allodynia [85, 87, 93] while certain chemokine knockout mice fail to develop somatic sensitivity [108, 109]. Oh and colleagues [87] published an early example of nociceptive chemokine function when they showed that intraplantar administration of various chemokines such as CXCL12, CCL5, and CCL22 induce mechanical hypersensitivity lasting for at least 3 h. Since then numerous studies have reported that either exogenous peripheral (e.g., intradermal) or central (e.g., intrathecal) chemokine application induces mechanical hypersensitivity and/or thermal hyperalgesia [88, 90, 92, 99, 110, 111]. Intrathecal administration of CCL2 can produce mechanical hypersensitivity within 30 minutes and hyperalgesic effects can be detected up to 4 days after administration [88]. In contrast, CCL2-induced thermal hypersensitivity resolves 24 h after administration [88]. Interestingly, transgenic mice lacking the CCR2 receptor (principle receptor for CCL2) are resistant to the development of mechanical hypersensitivity following mechanical nerve injury however, following complete Freund’s adjuvant- (CFA-) induced neural inflammation, these mice display only a small, insignificant decrease in mechanical sensitivity compared to control animals and show no changes in thermal nociception [108]. These data suggest specificity for chemokine function with respect to type of injury and pain modality.

Studies utilizing antagonists against chemokine signaling provide evidence for a therapeutic role with respect to neuropathic pain. In two different models of HIV-1 associated neuropathy, Bhangoo et al. [85, 91] demonstrate that antiretroviral drug- or viral coat protein-, gp120-, induced mechanical hypersensitivity is attenuated by acute, systemic treatment with CCR2 or CXCR4 antagonists. Other pain eliciting models such as focal demyelination, CFA-induced inflammation and sciatic nerve constriction have demonstrated the therapeutic effects of chemokine receptor antagonists [84, 111, 112].

4.5. Chemokines and Cystitis

Clinical studies assessing patients with various pelvic inflammatory/pain syndromes and rodent models of visceral inflammation indicate a role for chemokines in the initiation or maintenance of visceral inflammation. CYP-induced inflammation increases the expression of CXCL12/CXCR4, CX3CL1/CX3CR1, CCL2/CCR2, and CXCL1 in the urinary bladder and CCL2 and CXCL1 in urine [113–117]. Blockade of CXCL10 signaling reduces severity of CYP-induced bladder inflammation by reducing hyperplasia, epithelial erosions, and infiltration of T cells, mast cells, and killer T cells in the bladder urothelium of rats [118]. Additionally, elevated chemokines levels have been detected in the seminal plasma and peripheral immune cells of patients with pelvic inflammatory/pain syndromes such as ulcerative colitis, chronic prostatitis, chronic pelvic pain syndrome, and BPS/IC [118–120]. Bladders from patients with ulcerative BPS/IC have increased mRNA expression of CXCL9, CXCL10, and CXCL11 in the interstitium and CXCR3 in the urothelial membrane [121]. Both Tyagi et al. [122] and Corcoran et al. [123] detected elevated chemokines, specifically CXCL1, CXCL10, CXCL12- , and CCL7, in the urine of patients with ulcerative BPS/IC. Interestingly, CCL7 levels decreased following hydrostatic distention and were correlated with symptom relief [123]. Tyagi et al. [122] suggest the presence of urinary chemokines originates from bladder tissue because urinary CXCL10 levels are present at levels much higher than those detected in serum.

Considering the extensive data implicating a sensory and signaling role for the urothelium, it is possible that urothelial-derived chemokines, especially those detected in the urine of BPS/IC patients, contribute to symptoms of bladder dysfunction. Recently, the functional contribution of the urothelium has advanced beyond the view of a passive barrier and is now suggested to have “neuron-like” properties such as plasticity and sensory transduction capabilities, especially in the context of bladder inflammation [124, 125]. Functional receptor expression, in conjunction with secretion capabilities, allows the urothelium to respond to stimuli and reciprocally communicate with detrusor smooth muscle cells, suburothelial nerve plexus, or interstitial cells [126–130]. It is possible that chemokine signaling via receptor expression in urothelial cells may consequently activate downstream targets that promote either the transcription or the expression and release of other inflammatory mediators or excitatory amino acids. Urothelial derived mediators such as adenosine triphosphate or nitric oxide may then influence the suburothelial nerve plexus to affect micturition reflex function [129].

5. CXCL12 and CXCR4

Our lab examined the expression and therapeutic effect with receptor blockade of the chemokine CXCL12, and one of its two receptors, CXCR4, in a rodent model of cystitis. This chemokine/receptor pair was of interest because of its demonstrated role in visceral inflammation and pathology in other abdominopelvic organs. Mikami et al. [119] show that CXCR4 peripheral T-cell expression was increased in patients with ulcerative colitis and that expression levels correlated with disease activity. Additionally, chemically induced colitis in mice leads to an increase of CXCR4-positive leukocytes and CXCL12 expression in colonic tissue [119]. Administration of a CXCR4 antagonist reduced these inflammatory effects. To address the role of CXCL12/CXCR4 signaling in normal micturition and inflammation-induced bladder hyperreflexia, bladder inflammation in adult female Wistar rats was induced by injecting CYP intraperitoneally at acute (150 mg/kg 4 h), intermediate (150 mg/kg 48 h), and chronic (75 mg/kg every third day for 10 days) time points. CXCL12 and its receptor, CXCR4, were examined in the whole urinary bladder of control and CYP-treated rats using complementary approaches including enzyme-linked immunosorbent assays (ELISAs), qRT-PCR, and immunostaining techniques. ELISAs, qRT-PCR, and immunostaining experiments revealed a significant increase in CXCL12 and CXCR4 expression in the whole urinary bladder and particularly in the urothelium, with CYP treatment [114]. CXCL12/CXCR4 interactions in micturition were evaluated using conscious cystometry with continuous instillation of saline and CXCR4 receptor antagonist (AMD3100 5 μM) administration in control and CYP- (48 h) treated rats. Receptor blockade of CXCR4 using AMD3100 increased bladder capacity in control (no CYP) rats and reduced CYP-induced bladder hyperexcitability as demonstrated by significant increases in intercontraction interval, bladder capacity, and void volume [114]. In these studies, AMD3100 is most likely acting at the level of the urothelium for several reasons: (1) both mRNA and histologic analyses showed that the greatest expressional increase for both CXCL12 and CXCR4 following CYP treatment was in the urothelium (2) histologically, CXCR4 had a restricted presentation being expressed only in the urothelium in both control and CYP treated bladders (3) repeated attempts did not demonstrate CXCL12- or CXCR4-IR in the suburothelial nerve plexus [114]. These results suggest a role for CXCL12/CXCR4 signaling in both normal micturition and with bladder hyperreflexia following bladder inflammation.

6. CCL2/CCR2

The chemokine, CCL2 (monocyte chemoattractant protein-1, MCP), and its high-affinity receptor, chemokine (C–C motif) receptor 2 (CCR2), have been implicated in hypersensitivity following neuronal inflammation or mechanical injury [88, 92, 99, 101, 108, 131, 132] in the central (i.e., spinal cord) and peripheral (i.e., DRG) nervous system. Blockade of CCR2 reduces established pain behaviors resulting from chronic nerve injury [88, 99, 101, 132] and exogenous application of CCL2, either centrally or peripherally, can elicit exaggerated sensory behavioral responses in rodents [88, 92, 99, 101, 132]. In addition, CCR2 null mice fail to develop somatic sensitivity following partial sciatic nerve ligation [108] whereas mice with CCL2 overexpression in astrocytes develop exaggerated thermal hyperalgesia following complete Freund’s adjuvant-induced inflammation [131].

Our recent studies demonstrate novel findings with respect to the contribution of CCL2/CCR2 interactions with bladder inflammation-induced changes in bladder function and somatic sensitivity in female rats. We demonstrate that CYP-induced cystitis increases (1) CCL2 and CCR2 transcript and protein expression in the rat urinary bladder and (2) the number of bladder-associated CCR2-immunoreactive bladder afferent cells in the lumbosacral DRG [113]. Blockade of CCR2 receptor interactions with the highly selective receptor antagonist, RS504393 (5 μM), at the level of the urinary bladder, increased bladder capacity, decreased void frequency, and reduced somatic sensitivity of the hindpaw and pelvic region following CYP treatment [113]. These results extend previous findings [83, 88, 92, 133, 134] by demonstrating that CCL2/CCR2 interactions contribute to inflammation-induced bladder dysfunction and increased referred somatic sensitivity.

CCL2/CCR2 interactions at the level of the urothelium and suburothelial nerve plexus in the urinary bladder are likely to contribute to bladder dysfunction and increased somatic sensitivity following CYP-induced cystitis. Intravesical instillation of RS504393 likely makes direct contact with the urothelium that expresses CCR2 and the increased urothelial permeability due to CYP treatment makes it likely that intravesical RS504393 also contacts suburothelial nerves. Our studies did not differentiate between direct urothelial and nerve-mediated CCR2 effects versus indirect urothelial-mediated communication with the detrusor smooth muscle, suburothelial nerve plexus, and/or interstitial cells as previously suggested [126, 127]. It is possible that urothelial CCL2/CCR2 signaling facilitates the release of urothelial-derived mediators such as adenosine triphosphate or nitric oxide that may then influence underlying structures such as the suburothelial nerve plexus and/or detrusor smooth muscle [126, 127, 129].

Alternatively, or in addition to urothelial-mediated mechanisms, CCL2/CCR2 interactions in bladder associated DRG neurons may contribute to inflammatory-induced changes in bladder sensory physiology and function. CYP treatment triggered a robust increase in the percentage of bladder afferent cell bodies expressing CCR2-IR [113]. These results complement previous findings demonstrating an increase in the percentage of primary sensory afferent cells expressing CCL2 and/or CCR2 following focal nerve demyelination, sciatic nerve ligation, or chronic constriction injury [83, 84, 89, 92, 133, 135]. Jung and Miller [94] demonstrate that depolarization of cultured sensory neurons is sufficient to induce CCR2 mRNA expression suggesting that heightened sensory neuron activity during states of injury or inflammation may contribute to elevated levels of neuronal CCR2 expression. Increased receptor expression may explain why peripheral nerve damage or inflammation can also change the functional properties of sensory neuron populations such that an increasing percentage of DRG neurons responds to CCL2 application or neurons respond with increased intracellular calcium ion currents and/or frequency of EPSCs [82–84, 99, 101, 135]. Therefore, it is possible that CCL2 released, in vivo, by DRG neurons, glial cells, and urothelial cells could contribute to nociceptive sensations/behaviors by autocrine or paracrine signaling mechanisms.

7. Cytokines

In addition to the chemokine family, ample evidence suggests that other cytokines contribute to the development of hyperalgesia and allodynia following injury or inflammation [79, 136]. Cytokine receptors have been detected in neurons and glial cells, especially after peripheral neuropathy [136]. Cytokine/receptor interactions can activate signaling pathways that induce transcription and release of other proinflammatory/nociceptive mediators including NGF and other cytokines and chemokines from peripheral neurons or glial cells [95–97, 137]. Cultured human detrusor smooth muscle cells secrete low levels of cytokines (IL-6 and IL-8) and chemokines (CCL2 and CCL5) and exposure to the inflammatory cytokines, IL-1β and TNF- , increases this release [138, 139]. The expression of cytokines, alone or in combination with other cytokines, growth factors, or other mediators, may form a bidirectional communication network between the nervous system and the immune system [140].

Studies examining cytokine expression using a CYP model of cystitis have detected elevated IL-6, IL-1 , and IL-4, among others, protein and mRNA levels in the urine and urinary bladder [64, 115]. Cytokine transcription and expression increase in the urinary bladder of patients with ulcerative BPS/IC [121, 123, 141]. Certain cytokine mRNAs, including IL-6 and TNF- , have been detected in the interstitium and urothelium of these biopsies [121, 141]. Additionally, reports have repeatedly detected elevated IL-6 in the urine of patients with ulcerative BPS/IC. Increased levels have been suggested to indicate either severity of inflammation [39] or correlate with pain scores and nocturia [141, 142]. Lotz et al. [141] propose that the bladder is the primary source of urinary IL-6 because it was not detected in ureteral urine.

Recently we examined the expression and function of another cytokine, TGF-β, in the urinary bladder with inflammation. TGF-β has an extensive role in the immune system and has been implicated in nociception and detected in the urine and urothelium of rats treated with CYP-induced cystitis [143].

8. Transforming Growth Factor-Beta (TGF-

8.1. Background

The TGF-β superfamily is comprised of at least 35 pleiotropic proteins belonging to four subfamilies grouped by their sequence homology—decapentaplegic-Vg-related (DVR), activin/inhibin, TGF-β sensu stricto, and other divergent members [144]. Even though TGF-β superfamily members have distinct expression patterns and regulate a variety of functions, they are each translated as a preproprotein that contains a peptide sequence signaling to the endoplasmic reticulum, a N-terminal prodomain, and a C-terminal mature protein [144, 145]. After proteolytic processing and posttranslational modifications, the C-terminal fragment is either secreted as a mature protein dimer or forms a latent complex by maintaining a noncovalent bond to the prodomain [144, 145].

The canonical members of TGF-β sensu stricto are one such proprotein to form a latent complex. The interactions between the N-terminal prodomain, termed latency associated peptide (LAP), and the mature TGF-β dimer are sufficient to sequester its extracellular activity [146]. Additionally, LAP associates with a latent TGF-β binding protein (LTBP) that regulates TGF-β bioavailability by chaperoning the complex to the extracellular matrix [147]. The subsequent activation of latent TGF-β in the extracellular matrix via LAP cleavage occurs by protease-dependent or protease-independent (protons, integrins, reactive oxygen species, etc.) mechanisms [145, 148–151].

After its secretion, the mature or activated protein dimers process a signal through transmembrane Ser-Thr receptor kinases [144]. The TGF-β family of receptors is comprised of type I and type II receptors. Type II receptors selectively bind their respective ligands to define part of the specificity of signal transduction [144]. Ligand binding can either be “sequential” or “cooperative” and may involve an accessory receptor (type III) to enhance ligand presentation [152]. Following receptor-ligand interaction, the type II receptor forms a heterotetrameric complex with the type I receptor to transphosphorylate residues of the Gly-Ser (GS) box [152]. The activated type I receptors then phosphorylate Smad-dependent or Smad-independent substrates to regulate the transcription of target genes [153].

Smad proteins exist in three families: receptor-activated, common mediator, and inhibitory. Receptor-activated (R-) Smads dock onto type I receptors and are phosphorylated on distal serine residues following receptor activation [153]. Phosphorylated R-Smads dissociate from the receptor and interact with common mediator Smad4 [153]. The oligomeric R-Smad/Smad4 complex then translocates to the nucleus where it alters the transcription of target genes [153]. Type I receptors not only function through Smad signaling but may also directly activate Smad-independent pathways such as TGF-β-activated kinase 1 (TAK1), Ras, nuclear factor-κB (NF-κB), and the mitogen-activated protein kinase (MAPK) subfamily members [154–159]. The variety of direct and context-dependent downstream signaling pathways preserves the multifunctional role(s) of TGF-β superfamily ligands while providing the specificity required to control distinct target genes.

8.2. Immune Response

The canonical members of TGF-β sensu stricto maintain immunological function by regulating the initiation and resolution of the immune response and a comprehensive review has been previously published [160]. Briefly, activated TGF-β at the site of injury may initiate a proinflammatory milieu characterized by matrix remodeling and the recruitment and activation of leukocytes [160, 161]. TGF-β may then aid in resolving the primary immune response and support a milieu for tissue repair and immunological memory to progress by suppressing the proliferation, differentiation, and survival of a subset of lymphocytes [160].

To initiate an immune response, TGF-β may mobilize monocytes, mast cells, and granulocytes to the site of injury and influence their adhesion to the extracellular matrix [160, 162–164]. While TGF-β may also recruit monocyte-derived macrophages, their activation and function are typically inhibited to help resolve the immune response [161, 165, 166]. Since immune cells continue to infiltrate the site of injury, the extracellular matrix undergoes pathological remodeling characterized by protease secretion and matrix degradation [167]. TGF-β supports the remediation and repair of these tissues by increasing the deposition of matrix proteins and inhibiting protease activation [168].

To sustain the resolution of the immune response, TGF-β may regulate T-cell proliferation, differentiation, and survival [169]. TGF-β promotes T-cell growth arrest by suppressing interleukin-2 in areas of subthreshold antigen presentation [160, 170]. During the polarizing conditions of the immune response, TGF-β maintains peripheral immunological tolerance by inducing the transcription factor FoxP3 to promote CD4+ CD25+ T-cell differentiation to regulatory T cells [160, 171]. CD4+ T-cell differentiation to the T helper (Th) 1 and Th2 cell lineages, however, is inhibited by TGF-β mediated repression of the transcription factors T-bet and GATA-3, respectively [169, 172]. In addition to its effects on CD4+ T cells, TGF-β may also attenuate the cytotoxicity of CD8+ T cells by inhibiting its cytolytic genes [173].

TGF-β not only stabilizes T-cell expression and function to resolve the immune response but also regulates B-cell proliferation, survival, and development [174]. TGF-β inhibits both the proliferation and cell cycle progression of B cells through Smad-dependent or Smad-independent pathways [160, 175–177]. TGF-β utilizes comparable B-cell growth arrest pathways, as well as a distinct Smad-independent pathway, to induce the apoptosis of B cells [160, 178]. Lastly, TGF-β may regulate the maturation and activation of B cells through its induction of isotype switching, suppression of B-cell antigen receptor signaling, and inhibition of immunoglobulin secretion [160, 179, 180].

8.3. Nociception

The members of TGF-β sensu stricto contribute to both the peripheral and central processing of noxious stimuli. TGF-β1 and TGF-β2 have been demonstrated to increase de novo neuropeptide synthesis in the DRG that may directly sensitize primary afferent nociceptors [181, 182]. TGF-β may also influence DRG excitability by regulating several ion channels including the voltage-gated potassium (Kv) channel and TRPV-1. Application of recombinant TGF-β1 in vitro has been demonstrated to downregulate KCNA4 gene expression and decrease A-type Kv currents in primary DRG cultures [183]. Additionally, TGF-β1 Smad-independent signaling may phosphorylate TRPV-1 on Thr residues and potentiate capsaicin-evoked calcium influx in the DRG [184, 185]. The subsequent prolonged depolarization and an impaired repolarization may lead to an amplification of nociceptive transmission and CNS input.

Unlike its role in the periphery, TGF-β in the CNS appears to be neuroprotective by regulating neuronal and nonneuronal response to inflammatory injury [186]. Nonneuronal glial cells have recently been recognized to enhance the proinflammatory milieu and facilitate the central processing of nociception [187]. Activated TGF-β in the CNS may inhibit the proliferation and activation of these spinal glial cells to attenuate the induction of neuropathic pain [188–190]. TGF-β may further reduce excitatory synaptic transmission of second-order neurons by directly suppressing the proinflammatory milieu in the spinal cord [189]. As a result of its biphasic and modulatory role in the peripheral and central transmission of nociception, TGF-β appears to have a profound impact on the perception of pain and may initiate, in part, pathological pain syndromes.

8.4. Role(s) in Cystitis

TGF-β ligands and its cognate receptors are expressed at low, basal levels in rat urinary bladder tissues [191]. Following chemically (CYP) induced cystitis of varying durations, TGF-β ligand, and receptor expression appears to display a time- and tissue-dependent regulation. TGF-β exhibits a delayed, but sustained, increase in urinary bladder gene and protein expression 8–48 h after CYP treatment [143, 191, 192]. Furthermore, urinary excretion of active and latent TGF-β1 is increased up to 100-fold 24 h after acute CYP treatment [143]. The aforementioned regulation of TGF-β gene and protein expression has been suggested to be more pronounced in the afferent limb of the micturition reflex suggesting a possible role in the development of LUT symptoms [191]. Its role in micturition reflex dysfunction was confirmed following the pharmacological inhibition of aberrant TGF-β signaling with cystitis. Inhibition of TGF-β type I receptors 48 h after CYP-induced cystitis decreased urinary frequency and increased bladder capacity, void volume, and intercontraction intervals [191]. These studies raise the possibility of targeting TGF-β at the level of the urinary bladder to alleviate voiding dysfunction with cystitis.

9. Perspectives and Future Directions

Blockade of cytokine/receptor and chemokine/receptor signaling may represent a potential therapeutic target for inflammation-associated bladder dysfunction. In addition, the presence of certain inflammatory molecules in patient urine may be useful biomarkers for BPS/IC or other bladder disorders such as overactive bladder (OAB). Similar to BPS/IC, the etiology of OAB remains elusive however, based on patient biopsies an inflammatory contribution has been suggested [193–195]. Tyagi et al. [196] detected a 10-fold increase of CCL2 and the soluble fraction of the CD40 ligand (CD40L) in the urine of OAB patients versus controls. Various cytokines, epidermal growth factor (EGF), and the oncogene GRO-a were also elevated (3-5-fold) in the urine of OAB patients [196]. Whether certain inflammatory mediator/receptor interactions and downstream signaling pathways are redundant or unique across diverse bladder dysfunction or pelvic pain syndromes remains to be determined. Identification of urinary biomarkers in BPS/IC, OAB, or other bladder dysfunctions would improve diagnostic strategies and reduce invasiveness to the patient, improving exclusionary criteria, reducing time to diagnosis and aid in patient selection for pharmacological trials.

Abbreviations

BPS:Bladder pain syndrome
CCR2:Chemokine (C-C motif) receptor 2
CGRP:Calcitonin gene-related peptide
CNS:Central nervous system
CYP:Cyclophosphamide
DRG:Dorsal root ganglia
DVR:Decapentaplegic-Vg-related
EGF:Epidermal growth factor
ELISA:Enzyme-linked immunosorbent assay
EPSC:Excitatory postsynaptic current
FIC:Feline interstitial cystitis
GS:Glycine-serine
h:Hour(s)
HIV:Human immunodeficiency virus
IC:Interstitial cystitis
Kv:Voltage-gated potassium
L:Lumbar
LAP:Latency associated peptide
LTBP:Latent transforming growth factor-beta binding protein
LUT:Lower urinary tract
MAPK:Mitogen-activated protein kinase
MCP:Monocyte chemoattractant protein
NF-κB:Nuclear factor-kappa B
NGF:Nerve growth factor
NIDDK:National Institute of Diabetes and Digestive and Kidney Diseases
OAB:Overactive bladder
PAG:Periaqueductal gray
PMC:Pontine micturition center
PNS:Peripheral nervous system
PS:Protamine sulfate
qRT-PCR:Quantitative reverse transcriptase polymerase chain reaction
R-:Receptor-activated
S:Sacral
SP:Substance P
T:Thoracic
TAK1:Transforming growth factor-beta-activated kinase 1
TGF-β:Transforming growth factor-beta
Th:T helper
TRP:Transient receptor potential
V:Vanilloid.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors gratefully acknowledge the technical expertise and support provided by the VT Cancer Center DNA Analysis Facility. The authors also acknowledge the technical support and expertise of current laboratory members Dr. Beatrice Girard, Susan Malley, and Abbey Peterson in the execution of the described studies. This work was funded by National Institutes of Health (NIH) Grants DK051369 (MAV), DK060481 (MAV). Mr. Eric J. Gonzalez is supported by a supplement to DK051369. This publication was also supported by grants from the National Center for Research Resources (5 P30 RR 032135) and the National Institute of General Medical Sciences (8 P30 GM 103498) from the NIH.

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Copyright

Copyright © 2014 Eric J. Gonzalez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Study sheds new light on urinary tract infections in postmenopausal women

A UT Southwestern study suggests why urinary tract infections (UTIs) have such a high recurrence rate in postmenopausal women: several species of bacteria can invade the bladder walls.

UTI treatment is the most common reason for antibiotic prescriptions in older adults. Because of the prevalence of UTIs, the societal impact is high and treatment costs billions of dollars annually.

"Recurrent UTI (RUTI) reduces quality of life, places a significant burden on the health care system, and contributes to antimicrobial resistance," said Dr. Kim Orth, Professor of Molecular Biology and Biochemistry at UTSW and senior author of the study, published in the Journal of Molecular Biology.

The investigation demonstrates that several species of bacteria can work their way inside the human bladder's surface area, called the urothelium, in RUTI patients. Bacterial diversity, antibiotic resistance, and the adaptive immune response all play important roles in this disease, the study suggests.

"Our findings represent a step in understanding RUTIs in postmenopausal women," said Dr. Orth, also an Investigator of the prestigious Howard Hughes Medical Institute who holds the Earl A. Forsythe Chair in Biomedical Science and is a W.W. Caruth, Jr. Scholar in Biomedical Research at UTSW. "We will need to use methods other than antibiotics to treat this disease, as now we observe diverse types of bacteria in the bladder wall of these patients."

Since the advent of antibiotics in the 1950s, patients and physicians have relied on antibiotics for UTI treatment.

"As time went on, however, major antibiotic allergy and resistance issues have emerged, leading to very challenging and complex situations for which few treatment choices are left and one's life can be on the line," said Dr. Philippe Zimmern, Professor of Urology and a co-senior author. "Therefore, this new body of data in women affected by RUTIs exemplifies what a multidisciplinary collaboration can achieve going back and forth between the laboratory and the clinic."

UTIs are one of the most common types of bacterial infections in women, accounting for nearly 25 percent of all infections. Recurrence can range from 16-36 percent in premenopausal women to 55 percent following menopause. Factors thought to drive higher UTI rates in postmenopausal women include pelvic organ prolapse, diabetes, lack of estrogen, loss of Lactobacilli in the vaginal flora, and increased colonization of tissues surrounding the urethra by Escherichia coli (E. coli).

The latest findings build on decades of clinical UTI discoveries by Dr. Zimmern, who suggested the collaboration to Dr. Orth, along with other UT System colleagues.

The UTSW team, which included researchers from Molecular Biology, Pathology, Urology, and Biochemistry, examined bacteria in bladder biopsies from 14 RUTI patients using targeted fluorescent markers, a technique that had not been used to look for bacteria in human bladder tissue.

"The bacteria we observed are able to infiltrate deep into the bladder wall tissue, even past the urothelium layer," said first and co-corresponding author Dr. Nicole De Nisco, an Assistant Professor of Biological Sciences at UT Dallas who initiated this research as a postdoctoral fellow in Dr. Orth's lab. "We also found that the adaptive immune response is quite active in human RUTIs."

Accessing human tissue was key, the researchers note, as the field has largely relied on mouse models that are limited to lifespans of 1.3 to 3 years, depending on the breed.

"Most of the work in the literature has dealt with women age 25 to 40," said Dr. Zimmern, who holds The Felecia and John Cain Chair in Women's Health, recently established in his honor. "This is direct evidence in postmenopausal women affected with RUTIs, a segment of our population that has grown with the aging of baby boomers and longer life expectancy in women."

Future studies will focus on determining effective techniques to remove these bacteria and chronic inflammation from the bladder, finding new strategies to enhance immune system response, and pinpointing the various bacterial pathogens involved in RUTIs.

Other team members from UTSW include Dr. Marcela de Souza Santos, former Assistant Professor of Molecular Biology Dr. Jason Mull, former Assistant Professor of Pathology Luming Chen, a graduate student researcher in the Medical Scientist Training Program and Inkkaruch Kuprasertkul, a medical student who worked on the investigation as part of her summer research project. Dr. Kelli Palmer, Associate Professor of Biological Sciences at UTD and Cecil H. and Ida Green Chair in Systems Biology Science Fellow, and Michael Neugent, UTD doctoral candidate, also contributed.

The study was funded by the National Institutes of Health (NIH), The Welch Foundation, Once Upon a Time . and the Cecil H. and Ida Green Chair in Systems Biology Science.