Gerard Apodaca, PhD - US grants

The specialized umbrella cells lining the mucosal surface of the urinary bladder form a barrier between the urine and the underlying muscle layers and vasculature. When this barrier function is disrupted, disease may ensue (e.g. in interstitial cystitis or bacterial infection). The barrier function of the uroepithelium depends on several factors including: formation of a polarized epithelium with tight junctions, presence of an apical plasma membrane highly impermeable to water and urea, and a surface layer of glycosaminoglycans. Understanding how barrier function is disrupted in disease requires that normal barrier function and development of the uroepithelium be defined. As such, the first aim of this proposal is to characterize the development of polarity and uroepithelial phenotype in a newly-established primary uroepithelial cell culture model. These cultures obtain high transepithelial resistance (up to 10,000 omegacm2), and have apical sodium channel activity. Like umbrella cells found in vivo, cultured umbrella cells have an asymmetric unit membrane and express the AE-31 antigen and uroplakins. In addition, two aspects of umbrella cell barrier function that are only poorly understood will be examined. These include the requirement that the umbrella cell accommodate large changes in bladder volume - likely to be mediated by fusiform vesicles that are inserted/removed from the apical membrane in response to filling and emptying of the bladder - as well as the ability of these cells to limit the amount of apical endocytosis and prevent endocytosed urine from reaching the underlying tissue. The hypothesis that fusiform vesicle exocytosis is regulated by stretch and will be modulated by stretch-sensitive channels, secondary messenger cascades, and SNARE fusion complexes will be examined in Aim II. The effects of stretch receptor antagonists on fusiform vesicle exocytosis will be examined, as will modulators of [Ca2+]i, cAMP production, protein kinase C activation, and SNARE fusion proteins. In Aim III the following hypotheses will be tested: (1) apical endocytosis will be inhibited during periods of bladder filling; (2) endocytosis will be stimulated following voiding; and (3) internalized urine will be primarily recycled to the apical pole of the cell or be delivered to lysosomes. A combination of biochemical and morphological tools will be used to characterize the endocytic and postendocytic traffic of stretched and unstretched primary cultured and freshly isolated uroepithelium. The proposed experiments will provide novel insights into normal uroepithelium development and barrier function.

[unreadable] DESCRIPTION (provided by applicant): The uroepithelium, which lines the inner surface of the bladder, forms an impermeable barrier and also functions as an integral part of a sensory web. Through uroepithelial-associated channels and receptors, the uroepithelium receives sensory input such as changes in hydrostatic pressure and binding of mediators such as ATP. These input signals stimulate membrane turnover in the outermost umbrella cell layer and release of sensory output from the uroepithelium in the form of mediators that communicate changes in the uroepithelial milieu to the underlying tissues, altering the function of the bladder. Adenosine is a universally produced nucleoside that participates in the normal function of all organ systems, and preliminary data indicates that the uroepithelium is a site of adenosine biosynthesis and expresses all four adenosine receptors (A1, A2a, A2b, and A3). Adenosine binding to these receptors stimulates exocytosis in the umbrella cell layer. The goal of this proposal is to test the hypothesis that adenosine, acting through uroepithelial-associated adenosine receptors, functions as a sensory input molecule that stimulates membrane turnover in the umbrella cell layer and alters bladder function. The first aim of this proposal will define the mechanism and function of adenosine biosynthesis and turnover by the uroepithelium. Selective inhibitors of enzymes/transporters as well as knockout mice lacking expression of adenosine kinase and adenosine deaminase will be used to define the mechanisms of adenosine production and turnover by the uroepithelium. Adenosine and its metabolites will be measured with a novel, highly sensitive and specific 3D-ion trap HPLC-mass spectrometric method. The second aim will define if uroepithelial-associated adenosine receptors modulate bladder function. The Cre/loxP system will be used to generate mice lacking uroepithelial expression of A1 and A2a receptors. Bladder function will be studied in these mice as well as in knockout mice globally lacking A1, A2a, A2b, and A3 receptor expression in all tissues. The third aim will define the role of adenosine and adenosine receptors in modulating membrane turnover in umbrella cells. Selective agonists and knockout mice lacking uroepithelial or global expression of A1 and A2a receptors will be used to define if these receptors are important for regulating endocytosis/exocytosis in the uroepithelium. The fourth aim will explore which signaling pathways act downstream of adenosine receptors to modulate membrane turnover in the uroepithelium. A combined biochemical/pharmacological approach and permeabilized cell systems will be used to define which G proteins and second messenger pathways are involved in regulating membrane traffic in the umbrella cell layer. The results of these studies will increase our understanding of the role of adenosine and its receptors in the uroepithelial-associated sensory web, and will ultimately allow us to understand how perturbations in uroepithelial-associated receptor expression and signal output can contribute to bladder diseases such as interstitial cystitis and detrusor overactivity.Project Narrative. Adenosine is a natural product of cells that regulates the function of the cardiovascular, neuronal, and renal organ systems. This grant will explore if adenosine regulates the delivery and recovery of membrane at the free surface of the cells lining the bladder, and whether adenosine affects bladder filling and voiding. Understanding adenosine function is important because it will help us to understand how the cells lining the bladder can aid in regulating the function of the bladder and how to treat diseases like interstitial cystitis and bladder overactivity. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Umbrella cells line the inner surface of the urinary bladder, ureters, and renal pelvis, forming an impermeable barrier that separates the urinary space from the underlying muscle layer. These cells experience profound and reversible morphologic changes from a roughly inverted umbrella shape in empty bladders to one that is flat and squamous as the bladder fills, all the while maintaining a tight barrier to paracellular transport. The latter function is dependent on the tight junction (TJ), an intercellular multi-protin complex located at the upper-most portion of the lateral membrane of epithelial cells that mediates cell adhesion and modulates paracellular transport between the lumen and the underlying tissue. Claudins, a group of tetraspan membrane proteins, are structural and functional component of the TJ. Our preliminary results indicate that during experimental filling the umbrella cell TJ ring expands and the paracellular resistance of the uroepithelium drops significantly. These changes are reversed upon voiding. The central hypothesis of our proposal is that during bladder filling and voiding, insertion and removal of claudin-based pores accompany expansion and contraction of the TJ ring, respectively, and that one function of paracellular transport is to signal the degree of epithelial stretch to the cells underlying the uroepithelium. The first aim explores how TJ organization and claudin rearrangements promote increased paracellular permeability during bladder filling. Based on a mathematical model of the uroepithelium permeation pathways we will conduct measurements of the following parameters to determine their contribution to the decrease in paracellular permeability during filling: length of junction per unit area of the epithelium, resistance of the lateral space, TJ strand number, and junctional resistance during experimental filling in response to changes in claudin expression. For these studies we will combine in situ adenoviral transduction to deplete endogenous claudins or overexpress tagged-claudins in umbrella cells, biochemistry to assess changes in the expression of claudins at the TJs, and electrophysiology to determine changes in paracellular permeability. The second aim will investigate how the TJ accommodates cell shape changes during bladder filling and voiding. Experiments will examine the hypothesis that TJ ring expansion requires Rab13-regulated exocytosis, while ring contraction involves claudin endocytosis. To define the machinery that regulates TJ ring expansion and contraction during bladder filling and voiding we will use a combination of biochemistry, in situ adenoviral transduction, and morphology. The goal of the third aim is to elucidate the physiologic role of increased paracellular permeability during filling. Here, we will use in situ transduction of pore-forming or barrier-like claudins as well as pharmacological maneuvers to alter the paracellular permeability of the epithelium, and then assess how increased or reduced conductance across the TJs affects the function of tissues subjacent to the epithelium.

DESCRIPTION (provided by applicant): Congenital anomalies of the kidney and urinary tract (CAKUT) are developmental disorders that occur in 1 out of every 500 live births, yet the cellular and molecular basis of these malformations has not been revealed. UPK3a, which encodes the type I transmembrane protein uroplakin 3a (UPK3a), is one gene targeted in CAKUT. Substitution of a Pro residue for a Leu (P273L) in the cytoplasmic domain of UPK3a leads to renal adysplasia and other urinary tract defects. However, the function(s) of UPK3a during urinary tract development, and the reason why mutations in this protein lead to CAKUT are not known. Using zebrafish larvae, we find that loss of expression of the UPK3a ortholog Upk3l leads to urinary tract (i.e., pronephros) dysfunction as a result of altered expression of Par polarity complex proteins (Par3, Par6, aPKC?) and defects in ezrin activation and microvilli formation. Moreover, our recent genetic and biochemical studies demonstrate that UPK3a/Upk3l may exert its influence by binding to aPKC? and then by fostering interactions between aPKC? and ezrin at the apical pole of pronephric tubule epithelial cells. Based on these observations, we propose that urinary tract development is dependent on the ability of UPK3a to promote UEEC differentiation, and mutations in UPK3a (e.g., P273L) perturb this function, leading to CAKUT. In our first aim we will use biochemistry to further define the cytoplasmic motifs in UPK3a and Upk3l that bind to the Par complex proteins and vice-versa. We will also define whether recruitment of the Par complex to the apical membrane of pronephric tubule cells depends on interactions with UPK3a/Upk3l. Furthermore, we will ectopically express UPK3a/Upk3l in MDCK cells, which do not normally express these proteins, and assess what impact this has on Par complex recruitment. In the second aim we will define how ezrin interacts with UPK3a/Upk3l and whether this interaction is critical for microvilli formation. We will further define whether aPKC? is responsible for phosphorylating ezrin-T567, a critical step in ezrin activation. In the third aim we will use MDCK cells as a model system to explore whether the P273L mutation alters ER exit and apical surface delivery of UPK3a. We will also use biochemistry and NMR spectroscopy to define whether the C-terminal tail of UPK3a contains structure, whether this structure is perturbed by the P273L mutation, and whether this mutation affects interactions with its binding partners. The proposed work is important because it will provide fundamental new insights into the basic mechanisms of lower urinary tract development and epithelial differentiation, into the role of UPK3a in these processes, and into the molecular and cellular basis of CAKUT.

Lower urinary tract symptoms (LUTS), in particular storage symptoms (urinary incontinence) are a major health related problem in the elderly. Yet, there remains insufficient understanding of how aging alters normal bladder physiology, and how these changes contribute to the etiology of LUT disorders in the elderly. Much of research past and present, has focused on detrusor muscle function and changes in the central neurological control of aging-related LUT function; however, much less is known about the role of the urothelium (UT) in these events. While previously thought of as a simple barrier, the urothelium communicates with the CNS via a local urothelial-afferent signaling pathway. Our preliminary data show that aged UT has altered mitochondrial function, including increased production of reactive oxygen species (ROS), which we hypothesize leads to lysosomal dysfunction, altered release of mediators, and defects in UT-afferent signaling, culminating in abnormal urodynamic behavior. Thus, our overall hypothesis is that age-related changes in the UT and adjacent bladder wall result in a pro-aging cellular phenotype that disrupts UT-cell signaling resulting in abnormal urodynamic behavior in the elderly. Our multidisciplinary research team will elucidate the effect of aging and oxidative/lysosomal stress on urothelial physiology and the impact this has on cross talk between the UT and other cells within the bladder wall. Using an aging (3-30 mo) rat model, we will in Aim #1 define how changes in bioenergetics and oxidative stress impact urothelial aging by using functional assays to measure changes in both mitochondrial function and architecture. In Aim #2, we will determine how lysosomal dysfunction contributes to urothelial aging. Here we will use stereology as well as biochemical and morphological tools to examine why degradation and mitophagy are impaired in aging urothelium. In Aim #3, we will determine if increasing mitochondrial/lysosomal function will enhance UT-signaling and resultant bladder function. We will use a multi-disciplinary approach including measurement of transmitter release and sophisticated imaging techniques coupled with recording bladder afferent nerve activity to examine how aging and increased mitochondrial oxidative stress alters UT- cell communication. In each aim, we will also examine whether treatments (mitotempo; metformin) that reduce oxidative stress/lysosome dysfunction can improve urothelial (and in vivo bladder function) in aged rats. In sum, our intriguing preliminary data combined with our extensive expertise and resources places our research team in a unique position to examine how direct and indirect factors promote UT dysfunction in bladder aging.

Abstract: During filling and voiding, the degree of tension in the bladder wall must be sensed and then relayed to the central nervous system, otherwise bladder dysfunction ensues. While there is a general understanding of the role of sensory neurons (i.e., wall mechanoceptors) in these events, we have fewer insights into how non-neuronal tissues contribute to tension sensation and transduction in the bladder. Our studies focus on the urothelium, which forms the direct interface between the bladder wall and the urinary space. This tissue responds to changes in tension by modulating its ion transport, membrane traffic, and release of mediators, which are hypothesized to alter bladder function, in part via a local urothelial:afferent reflex. However, we still have limited insights into how tension in the plasma membrane of urothelial cells is sensed, what is the nature of the downstream pathways that are activated in response to stretch, or how do these events contribute to bladder function and dysfunction. We hypothesize that urothelial-expressed PIEZO channels act as mechanosensors that in response to bladder filling promote Ca2+ entry, mediator release, and signaling to afferent nerve processes, promoting normal bladder function. We further hypothesize that dysregulation of PIEZO-dependent mechanotransduction in the urothelium will lead to bladder dysfunction. In Specific Aim 1, we will determine if PIEZO channels act as bona fide mechanosensors by demonstrating the following: (i) that functional PIEZO channels are expressed at the surface of urothelial cells; (ii) that urothelial expressed PIEZO channels respond to physiologically relevant stimuli (i.e., bladder filling); (iii) that PIEZO channels are required for mechanically regulated events including membrane traffic and mediator release; (iv) and that expression of loss-of-function or gain-of-function PIEZO mutants will lead to altered urothelial responses. In Specific Aim 2, we seek to understand how PIEZO channels promote urothelial:afferent signaling. Using novel tools, including ex vivo bladder imaging, we will explore the mechanisms by which PIEZO channels stimulate increases in intracellular Ca2+ ([Ca2+]i). Because PIEZO channels rapidly inactivate, there is likely to be a mechanism to amplify the original signal. Thus, we will define whether Ca2+-induced Ca2+ release or membrane depolarization act downstream of PIEZO channels to increase [Ca2+]i. We will also determine if the PIEZO-triggered rise in [Ca2+]i is critical for stimulating exocytosis and mediator release in the urothelium, and if urothelial PIEZO channels modulate urothelial:afferent signaling. In Specific Aim 3, we will begin to explore whether urothelial PIEZO channels contribute to bladder function and/or dysfunction. Specifically, we will determine whether loss of PIEZO expression or function results in bladder underactivity and if PIEZO channels with gain-of-function mutations lead to bladder overactivity. Finally, we will determine whether urothelial-expressed PIEZO channels contribute to the bladder hyperreflexia and pelvic allodynia associated with cyclophosphamide-induced cystitis.

Abstract: A critical component of the umbrella cell barrier is the apical junctional complex (AJC), a multipartite, belt-like structure comprised of the tight junction, the adherens junction, desmosomes, and an associated cytoskeleton. Functions of the AJC include regulation of paracellular flux, cell-cell adhesion, and mechanotransduction. Despite evidence that the umbrella cell AJC is integral to urothelial function and disrupted in several lower urinary tract disorders, we have limited understanding of key aspects of umbrella cell AJC biology and pathobiology including: (i) how the AJC maintains its continuity in the face of cyclical bladder filling and voiding; (ii) how the AJC is organized to undergo these transitions and the function of the cytoskeleton in these events; and (iii) how the umbrella cell AJC senses tension and whether pathologically high intravesical pressures stimulate AJC-associated mechanotransduction pathways. Our preliminary studies include the novel findings that during bladder filling the AJC perimeter expands dramatically, a process that depends on changes in the actin cytoskeleton and vesicular traffic, likely directed toward the AJC. In contrast, the AJC contracts soon after bladder voiding, events driven by the non-muscle myosin II-triggered contraction of the actin cytoskeleton, RhoA, as well as endocytosis. Based on available data, we hypothesize that critical functions of the umbrella cell AJC are to maintain urothelial barrier function by undergoing dynamic expansion and contraction and to serve as a site of mechanotransduction under normal and pathological conditions. To test this global hypothesis, we propose the following experiments. In Aim 1, we will use a newly developed biaxial stretching device, coupled with live-cell image analysis, to determine if increased strain triggers exocytosis of junction-associated proteins directed toward the AJC, and if release of strain stimulates their endocytosis. We will also assess if blocking AJC expansion perturbs urothelial barrier function. In Aim 2, we will focus on deciphering the function and organization of the umbrella cell AJC-associated cytoskeleton. We will use super-resolution confocal imaging, as well as electron microscopy to reconstruct the umbrella cell AJC in 3D. In addition, we will determine if formins drive actin polymerization in response to filling. In Aim 3, we will use tension sensors to determine if transmembrane proteins associated with the umbrella cell AJC sense force, and assess whether junction-associated signaling pathways are activated in response to partial bladder outlet obstruction (PBOO). Upon completion of these studies we will have new insights into how umbrella cell AJC dynamics contribute to urothelial barrier function, the organization of the AJC and the function of its associated cytoskeleton, and important new information about how the AJC senses and responds to perturbations in its mechanical milieu, including in response to PBOO.