Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

Location of corneal epithelial stem cells.

The longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia.

Hypercompliant apical membranes of bladder umbrella cells.

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder.

Generation of divergent uroplakin tetraspanins and their partners during vertebrate evolution: identification of novel uroplakins.

BACKGROUND: The recent availability of sequenced genomes from a broad array of chordates (cephalochordates, urochordates and vertebrates) has allowed us to systematically analyze the evolution of uroplakins: tetraspanins (UPK1a and UPK1b families) and their respective partner proteins (UPK2 and UPK3 families). RESULTS: We report here: (1) the origin of uroplakins in the common ancestor of vertebrates, (2) the appearance of several residues that have statistically significantly positive dN/dS ratios in the duplicated paralogs of uroplakin genes, and (3) the existence of strong coevolutionary relationships between UPK1a/1b tetraspanins and their respective UPK2/UPK3-related partner proteins. Moreover, we report the existence of three new UPK2/3 family members we named UPK2b, 3c and 3d, which will help clarify the evolutionary relationships between fish, amphibian and mammalian uroplakins that may perform divergent functions specific to these different and physiologically distinct groups of vertebrates. CONCLUSIONS: Since our analyses cover species of all major chordate groups this work provides an extremely clear overall picture of how the uroplakin families and their partner proteins have evolved in parallel. We also highlight several novel features of uroplakin evolution including the appearance of UPK2b and 3d in fish and UPK3c in the common ancestor of reptiles and mammals. Additional studies of these novel uroplakins should lead to new insights into uroplakin structure and function.

Uroplakins do not restrict CO2 transport through urothelium.

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (∼1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (∼7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (∼ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.

PKM2 Is Essential for Bladder Cancer Growth and Maintenance.

Pyruvate kinase M2 (PKM2) has been shown to promote tumorigenesis by facilitating the Warburg effect and enhancing the activities of oncoproteins. However, this paradigm has recently been challenged by studies in which the absence of PKM2 failed to inhibit and instead accelerated tumorigenesis in mouse models. These results seem inconsistent with the fact that most human tumors overexpress PKM2. To further elucidate the role of PKM2 in tumorigenesis, we investigated the effect of PKM2 knockout in oncogenic HRAS-driven urothelial carcinoma. While PKM2 ablation in mouse urothelial cells did not affect tumor initiation, it impaired the growth and maintenance of HRAS-driven tumors. Chemical inhibition of PKM2 recapitulated these effects. Both conditions substantially reduced complex formation of PKM2 with STAT3, their nuclear translocation, and HIF1α- and VEGF-related angiogenesis. The reduction in nuclear STAT3 in the absence of PKM2 also correlated with decreased autophagy and increased apoptosis. Time-controlled, inducible PKM2 overexpression in simple urothelial hyperplasia did not trigger tumorigenesis, while overexpression of PKM2, but not PKM1, in nodular urothelial hyperplasia with angiogenesis strongly accelerated tumorigenesis. Finally, in human patients, PKM2 was overexpressed in low-grade nonmuscle-invasive and high-grade muscle-invasive bladder cancer. Based on these data, PKM2 is not required for tumor initiation but is essential for tumor growth and maintenance by enhancing angiogenesis and metabolic addiction. The PKM2-STAT3-HIF1α/VEGF signaling axis may play a critical role in bladder cancer and may serve as an actionable therapeutic target. SIGNIFICANCE: Genetic manipulation and pharmacologic inhibition of PKM2 in mouse urothelial lesions highlight its essential role in promoting angiogenesis and metabolic addiction, events indispensable for tumor growth and maintenance.

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes.

The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation.

Bacteria-induced uroplakin signaling mediates bladder response to infection.

Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli.

Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution.

Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks.

Dominant role of CDKN2B/p15INK4B of 9p21.3 tumor suppressor hub in inhibition of cell-cycle and glycolysis.

Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15INK4B, p14ARF and p16INK4A), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15INK4B is markedly stronger than p16INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15INK4B, but not those expressing HRas and lacking p16INK4A, develop early-onset bladder tumors. The potency of CDKN2B/p15INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15INK4B on cell proliferation, and unveil mechanisms whereby p15INK4B aberrations may underpin cancer and non-cancer conditions.

Temporally and spatially controllable gene expression and knockout in mouse urothelium.

Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems.

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis.

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity--a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function.

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction.

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement.

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia.

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting.

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.

Uroplakins in urothelial biology, function, and disease.

Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder, and prostatic urethra. Although morphologically similar, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition, and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies urine composition. Urothelial cells express a number of ion channels, receptors, and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade the bladder mucosa, but also provide a route by which the bacteria ascend through the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis, and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins' biochemistry, structure, assembly, intracellular trafficking, and their emerging roles in urothelial biology, function, and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases.

The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis.

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.

Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly.

Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly.

Alterations in bladder function associated with urothelial defects in uroplakin II and IIIa knockout mice.

AIMS: The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/dysfunction were studied in male and female wild type and knockout (KO) mice. METHODS: UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types. RESULTS: These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO's, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former. CONCLUSIONS: These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction.