The Urothelium: Life in a Liquid Environment

Abstract

The urothelium, which lines the renal pelvis, ureters, urinary bladder, and proximal urethra, forms a high-resistance but adaptable barrier that surveils its mechanochemical environment and communicates changes to underlying tissues including afferent nerve fibers and the smooth muscle. The goal of this review is to summarize new insights into urothelial biology and function that have occurred in the past decade. After familiarizing the reader with key aspects of urothelial histology, we describe new insights into urothelial development and regeneration. This is followed by an extended discussion of urothelial barrier function, including information about the roles of the glycocalyx, ion and water transport, tight junctions, and the cellular and tissue shape changes and other adaptations that accompany expansion and contraction of the lower urinary tract. We also explore evidence that the urothelium can alter the water and solute composition of urine during normal physiology and in response to overdistension. We complete the review by providing an overview of our current knowledge about the urothelial environment, discussing the sensor and transducer functions of the urothelium, exploring the role of circadian rhythms in urothelial gene expression, and describing novel research tools that are likely to further advance our understanding of urothelial biology.

Keywords: bladder; epithelium; renal pelvis; ureter; urethra; urothelium.

Graphical abstract

FIGURE 1.

Cell layers of the urothelium. A: tissue organization of mouse bladder urothelium visualized using transmission electron microscopy. The urothelium comprises a superficial umbrella cell layer, 1–2 layers of intermediate cells, and a basal cell layer. The image is false-colored to highlight the following cell types: BC, basal cells; bIC, binucleate intermediate cell; Cp, capillary; IC, intermediate cells; Pe, pericyte; UC, umbrella cells. B: en face view of umbrella cell (UC) layer. Three umbrella cells are false-colored to reveal their relationship to one another. The boxed region is magnified in the inset image. Inset: arrowheads label the zippered apical membrane that lies directly above the tight junction. Microplicae (Mp) are marked. C: scanning electron micrograph of a dislodged umbrella cell revealing the tops of newly exposed intermediate cells (labeled with closed white circles). (Electron microscope images provided by Wily G. Ruiz.)

FIGURE 2.

Hinges, plaques, and discoidal and/or fusiform-shaped vesicles (DFVs) at the apical pole of umbrella cells revealed by transmission electron microscopy (TEM). A: ultrastructure of the apical region of a mouse umbrella cell. The position of “hinges” (cross sections through microplicae) are marked with arrowheads, and the location of intervening plaque regions (Pl) are indicated by thin blue-colored lines. (Image provided by Wily G. Ruiz.) B: morphology of discoidal-shaped vesicles (DV) in rat umbrella cells. C: TEM of the apical surface of a mouse umbrella cell. The boxed region is magnified in the inset. The asymmetric unit membrane (AUM) in the plaque region is marked. Note the presence of a glycocalyx on the luminal surface of the plasma membrane. (Image provided by Steven Truschel.) D: freeze, deep-etch electron microscopy reveals the architecture of plaque regions (Pl) and associated hinges (Hi). The inset is a magnified view of the 16-nm AUM particles that make up the plaque regions. [Image, provided by Dr. John Heuser, is used by permission from Wiley-Blackwell and is from Apodaca (32).] E: cartoon depicting the generalized architecture of the uroplakins embedded in a lipid bilayer. Blue circles on the luminal side of the uroplakins are sites of N-linked glycosylation. F: arrangement of uroplakins in the 16-nm AUM particles. Note that UPK1A/UPK2 heterodimers comprise the inner “ring” of the AUM particles, while UPK3A/UPK1B heterodimers form the outer ring. Interactions between UPK2 and UPK3A bridge the two regions.

FIGURE 3.

Location of urothelium in the lower urinary tract. Cartoon depicting the organization of the urothelium and underlying tissues in the renal pelvis, ureter, bladder, and proximal urethra. AT, adipose tissue; C, kidney cortex; D, detrusor; Fx, fornix; IM, inner medulla of kidney; IS, inner stripe, outer medulla of kidney; LP, lamina propria; M, mesangial cell layer; OS, outer stripe, outer medulla of kidney; Rs, rhabdosphincter; SE, simple epithelium; SkM, skeletal muscle layers; SM, smooth muscle layers; UO, ureteral orifices; Ut, urothelium. [Adapted from Dalghi et al. (156) and redrawn by Dennis R. Clayton.]

FIGURE 4.

Differentiation markers of the adult mouse urothelium. The differentiation markers expressed by the superficial (umbrella) cell layer, the intermediate cell layers, and the basal cell layer of the mouse urothelium.

FIGURE 5.

Development of the mouse urothelium. The cell types and differentiation markers expressed at embryo stage (E)12, E13, E15–17 of the developing mouse embryo and adult urothelium. The phenotypes of the associated cell types are presented in the bottom half of the figure. BC, basal cell; IC, intermediate cells (binucleate and mononucleate); P-0, early urothelial progenitor cell; SC, superficial cell; UC, umbrella cell.

FIGURE 6.

Regeneration of the mouse urothelium in response to injury. Left: urothelial response to acute injury. The loss of binucleate (4n+4n) umbrella cells, and/or subjacent cell layers, results in proliferation of the urothelium and regeneration of the superficial umbrella cell layer. This process depends on uroplakin⁺ intermediate cells, which undergo incomplete cytokinesis to generate binucleate (2n+2n) intermediate cells (bIC). In turn, the bIC give rise to superficial umbrella cells, which increase their ploidy by way of endoreplication. Right: urothelial response to serial and/or major injury. In the absence of bIC and their progenitors, KRT5⁺ basal cell, and possibly KRT5⁺, KRT14⁺-derived basal cells, give rise to all of the cell types, including the bICs. The latter give rise to superficial cells, which further differentiate into umbrella cells. BC, basal cell; IC, intermediate cells (binucleate and mononucleate); SC, superficial cell; UC, umbrella cell.

FIGURE 7.

Components of the umbrella cell junctional complex. A: the junctional complex is comprised of the belt-like tight junction, the belt-like adherens junction, and desmosomes, which form spot-like junctions. [Redrawn from Apodaca and Gallo (34).] B: transmission electron micrograph of mouse umbrella cell junctional complex (JC). AJ, adherens junction; De, desomosome; TJ, tight junction. Inset: higher magnification view of umbrella cell tight junction, showing “kissing points.” C: freeze fracture replica of apical tight junction-associated strands from frog bladder epithelium. [From Claude and Goodenough (140), with permission from Rockefeller Press.] D: three-dimensional reconstruction of rat umbrella cell layer immunolabeled with antibodies to CLDN8 and co-stained with TRITC-phalloidin (actin). The 3D image was tilted using Volocity software. One scale bar unit = 12.3 µm. [From Acharya et al. (4).] E: model of claudin architecture. The extracellular segments (ECS-1/ECS-2) are folded into five β-strands, which are numbered. The transmembrane domains are labeled I–IV. The red bar between β-strands three and four is a disulfide bridge. The position of the conserved claudin W-G-L-W-R signature motif is marked. ECH, short extracellular helix, which promotes cis-interactions between claudins on the same membrane domain. F: structure of the CLDN15 protomer. The ECH α-helix is colored cyan, and the β-strands formed by ECS1/2 are colored navy blue (β-strands are numbered). The α-helical transmembrane domains are colored white, and the approximate position of the lipid bilayer is indicated by the transparent blue box. G and H: two stacked rows of claudin polymers arranged in an anti-parallel fashion and viewed en face (G) or in cross section (H). When ECS1/ECS2 from four claudin protomers (colored in red, green, yellow, and blue) interact on adjacent membranes, they form pore-like, β-barrel channels. The location of the paracellular pores is indicated by red asterisks in G, and the red arrow in H indicates the pathway for ion flow through the pore formed by stacked, anti-parallel claudin protomers. F–H were generated using the Swiss PDB viewer software.

FIGURE 8.

Exocytic and endocytic traffic in umbrella cells. A: discoidal and/or fusiform-shaped vesicles (DFVs) and their associated apical cargoes, including uroplakins (UPKs), are assembled in the trans-Golgi network. There appear to be two populations of DFVs: one that is RAB11A/RAB8A⁺ and another one that is RAB27B⁺. Whether DFVs emerge presorted into distinct RAB11A ⁺ or RAB27B⁺ populations of vesicles (option 1), or emerge as a single population that subsequently undergoes sorting (option 2) is unknown. DFVs are transported to the apical pole of the umbrella cell, which is driven by the myosin motor proteins MYO5A and MYO5B. Although not shown, RAB11A/RAB8A can also recruit MYO5A. The final step in exocytosis, fusion, is mediated by SNAREs. While SNAP23 is a likely t-SNARE, the exact identity of the syntaxin isoform(s) involved is unknown. Possible candidates include STX1A, STX2, STX3, and STX1, and these may differ depending on the vesicle pool with which they interact. Those syntaxins not engaged in fusion complexes exist in an autoinhibited conformation, which results from interactions between the molecules NH₂-terminal H_abc, α-helical region, and the COOH-terminal half of the molecule. For clarity, DFV-associated SNAP23 and cognate syntaxins are not shown, but both are known to be localized to vesicles (presumably in an inactive conformation). There are several potential v-SNAREs including VTI1B, VAMP7, and VAMP8. The latter has been shown to cause accumulation of subapical DFVs in mouse knockout models (751), and is indicated as being associated with DFVs. [Adapted from Gallo et al. (237).] B: localization of endocytosed wheat germ agglutinin-FITC (green) and TJP1 (red) in umbrella cells. TJP1 marks the tight junction, but is also associated with the wheat germ-labeled endosomes in response to voiding. [From Khandelwal et al. (361), with permission from EMBO Press.] C: cationized ferritin was instilled into the bladder lumen, and the tissue fixed and processed for TEM after voiding. [From Khandelwal et al. (361), with permission from EMBO Press.] D: endocytic organelles in the rat umbrella cell. EL, endolysosome; ILV, intraluminal vesicle; LYS, lysosome; MVB, multivesicular body. [From Truschel et al. (703), with permission from the Public Library of Science.] E: model for regulation of apical endocytosis in umbrella cells. In response to basolateral stretch (arising from filling-induced distension or voiding-induced refolding), integrins signal through phosphatidylinositol 3-kinase (PI3K) and focal adhesion kinase (PTK2) to stimulate RHOA activity, possibly by activating the RHOA GEF, ARHGEF28. RHOA likely stimulates actin polymerization, which helps drive apical endocytosis. RHOA is inactivated by a GAP, possibly ARHGAP26, which along with endophilins may promote formation of apical endocytic vesicles. The final step, fission, is stimulated by DNM2. A small fraction of endocytosed apical membrane (and a small amount of internalized fluid) may be recycled, but the majority appears to be delivered to lysosomes by way of the MVB. The urothelial-specific protein SNX31 promotes the incorporation of uroplakins into ILVs.

FIGURE 9.

Expansion and contraction of the umbrella cell apical junctional complex. A: rats were catheterized and their bladders filled (fill), or filled and voided (void), or never allowed to fill (quiescent). The animals were perfusion fixed, and the distribution of CDH1, actin, and nuclei was assessed. B: upon bladder filling, the umbrella cell apical junctional complex (AJC) expands in a process that requires formin-dependent actin polymerization and RAB13-dependent exocytosis, likely of tight junction- and adherens junction-associated proteins. Upon voiding, the apical junctional complex contracts in a process that requires non-muscle myosin IIA (NMMIIA)-dependent constriction of the junction-associated actomyosin cytoskeleton and DNM2-dependent endocytosis of junction-associated proteins. [From Eaton et al. (192), with permission from American Society for Cell Biology.]

FIGURE 10.

The urothelium-associated sensory web. The function of the sensory web is to allow bidirectional communication between the urothelium and underlying tissues, in this case, sensory afferent termini. Input pathways expressed by the urothelium include channels that respond to stretch (step 1), or channels and receptors that are stimulated by mediators present in urine (step 2A), released apically from the umbrella cell layer (step 2A, and which promotes autocrine signaling), or released by afferent nerve termini (step 2B). Activation of urothelial-associated input pathways stimulates outputs in the form of mediators that are released apically (step 3A) or basolaterally (step 3B). The mediators released from the serosal surfaces of the urothelium bind to receptors/channels on afferent nerve termini, signaling the current physiochemical status of the urothelium to the central nervous system (CNS) (steps 4 and 5). The model presented focuses on the interaction between the urothelium and afferent nerve termini; however, similar pathways likely exist between the urothelium and interstitial cells, and between the urothelium and smooth muscle cells.

FIGURE 11.

The urothelium and subjacent tissues in the lower urinary tract. Tissues present in the walls of the renal pelvis, ureters, bladder, and proximal urethra include the urothelium, the lamina propria, a muscularis externa comprised of smooth muscle, and either a serosa (comprised of mesothelial cells) or an adventitia comprised of connective tissue components (not shown). BM, basement membrane; BV, blood vessel; IsC, interstitial cell; ImC, immune cell; LP, lamina propria; Me, mesothelium; ME, muscularis externa; MM, muscularis mucosae; Se, serosa; Ut, urothelium. (Cartoon created by Dennis R. Clayton.)

FIGURE 12.

Basement membrane and intraepithelial afferent nerve termini revealed by transmission electron microscopy (TEM). A: components of the mouse urothelial basement membrane (BM) include the basal lamina, which is comprised of the lamina lucida (LL) and lamina densa (LD), and the reticular lamina. The latter is chiefly comprised of fibrillar collagens. BC, basal cell. B: what is likely an individual afferent nerve fiber (false-colored in green) runs along the serosal surface of a mouse urothelial basal cell (BC). The lamina densa is false-colored blue. An intraepithelial nerve terminus (boxed), located within the basement membrane of the basal cell, is magnified in the inset. The nerve terminus includes a clathrin-coated vesicle (CCV), numerous small clear vesicles (CV), and dense-core vesicles (DCV). An interstitial cell (IntC) is seen below the nerve fiber. (TEM images provided by Steven Truschel.)

FIGURE 13.

Suburothelial interstitial cells. A: transmission electron microscopy (TEM) of mouse urothelium and subjacent interstitial cells. (TEM image provided by Steven Truschel.) B: localization of platelet-derived growth factor receptor-α (PDGFRA), actin, and nuclei (Nuc) in the mouse mucosa. PDGFRA⁺ interstitial cells reside below the urothelium (shaded blue in the left-most panel).

FIGURE 14.

Summary of urothelial input and output pathways. Mediators released by the urothelium (i.e., “outputs”) are indicated in blue text, and hypothesized target tissues for these outputs are indicated by blue arrows. Mediators present in the urinary space, or those released from afferent nerve termini, are marked in green text and serve as inputs, triggering urothelial responses. Putative input pathways present in the urothelium are indicated in green text and include numerous channels and receptors. There is limited understanding of the mediators released by interstitial cells or smooth muscle cells that impinge on urothelial function, thus the question marks. The purple arrow is a hypothetical mechanism whereby mediators released from one urothelial cell type can act as inputs in a different urothelial cell type.

FIGURE 15.

Examples of methods to study the urothelium. A: mouse urothelium transduced with an adenovirus encoding the Ca²⁺ sensor GCAMP5G (10). The entire mouse urothelium is transduced, but no other cell type in the bladder wall expresses the transgene, including subjacent interstitial cells. BC, basal cell; IC, intermediate cell; UC, umbrella cell. B: a “peeled” mouse bladder, connected to a catheter, is filled by a syringe attached to a Luer fitting. C: localization of UPK3A, KRT5, and actin in a peeled mouse bladder. Note that the thickness of the preparation is ~75 µm.