Expression and localization of the mechanosensitive/osmosensitive ion channel TMEM63B in the mouse urinary tract.

The epithelial cells that line the kidneys and lower urinary tract are exposed to mechanical forces including shear stress and wall tension; however, the mechanosensors that detect and respond to these stimuli remain obscure. Candidates include the OSCA/TMEM63 family of ion channels, which can function as mechanosensors and osmosensors. Using Tmem63bHA-fl/HA-fl reporter mice, we assessed the localization of HA-tagged-TMEM63B within the urinary tract by immunofluorescence coupled with confocal microscopy. In the kidneys, HA-TMEM63B was expressed by proximal tubule epithelial cells, by the intercalated cells of the collecting duct, and by the epithelial cells lining the thick ascending limb of the medulla. In the urinary tract, HA-TMEM63B was expressed by the urothelium lining the renal pelvis, ureters, bladder, and urethra. HA-TMEM63B was also expressed in closely allied organs including the epithelial cells lining the seminal vesicles, vas deferens, and lateral prostate glands of male mice and the vaginal epithelium of female mice. Our studies reveal that TMEM63B is expressed by subsets of kidney and lower urinary tract epithelial cells, which we hypothesize are sites of TMEM63B mechanosensation or osmosensation, or both.

Expression of Transgenes in Native Bladder Urothelium using Adenovirus-Mediated Transduction.

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA+ interstitial cells are fibroblasts.

Fibroblasts are crucial to normal and abnormal organ and tissue biology, yet we lack basic insights into the fibroblasts that populate the bladder wall. Candidates may include bladder interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells), which express the fibroblast-associated marker PDGFRA along with VIM and CD34 but whose form and function remain enigmatic. By applying the latest insights in fibroblast transcriptomics, coupled with studies of gene expression, ultrastructure, and marker analysis, we observe the following: 1) that mouse bladder PDGFRA+ cells exhibit all of the ultrastructural hallmarks of fibroblasts including spindle shape, lack of basement membrane, abundant endoplasmic reticulum and Golgi, and formation of homotypic cell-cell contacts (but not heterotypic ones); 2) that they express multiple canonical fibroblast markers (including Col1a2, CD34, LY6A, and PDGFRA) along with the universal fibroblast genes Col15a1 and Pi16 but they do not express Kit; and 3) that PDGFRA+ fibroblasts include suburothelial ones (which express ACTA2, CAR3, LY6A, MYH10, TNC, VIM, Col1a2, and Col15a1), outer lamina propria ones (which express CD34, LY6A, PI16, VIM, Col1a2, Col15a1, and Pi16), intermuscular ones (which express CD34, VIM, Col1a2, Col15a1, and Pi16), and serosal ones (which express CD34, PI16, VIM, Col1a2, Col15a1, and Pi16). Collectively, our study revealed that the ultrastructure of PDFRA+ interstitial cells combined with their expression of multiple canonical and universal fibroblast-associated gene products indicates that they are fibroblasts. We further propose that there are four regionally distinct populations of fibroblasts in the bladder wall, which likely contribute to bladder function and dysfunction.NEW & NOTEWORTHY We currently lack basic insights into the fibroblasts that populate the bladder wall. By exploring the ultrastructure of mouse bladder connective tissue cells, combined with analyses of their gene and protein expression, our study revealed that PDGRA+ interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells) are fibroblasts and that the bladder wall contains multiple, regionally distinct populations of these cells.

The molecular chaperone GRP170 protects against ER stress and acute kidney injury in mice.

Molecular chaperones are responsible for maintaining cellular homeostasis, and one such chaperone, GRP170, is an endoplasmic reticulum (ER) resident that oversees both protein biogenesis and quality control. We previously discovered that GRP170 regulates the degradation and assembly of the epithelial sodium channel (ENaC), which reabsorbs sodium in the distal nephron and thereby regulates salt-water homeostasis and blood pressure. To define the role of GRP170 - and, more generally, molecular chaperones in kidney physiology - we developed an inducible, nephron-specific GRP170-KO mouse. Here, we show that GRP170 deficiency causes a dramatic phenotype: profound hypovolemia, hyperaldosteronemia, and dysregulation of ion homeostasis, all of which are associated with the loss of ENaC. Additionally, the GRP170-KO mouse exhibits hallmarks of acute kidney injury (AKI). We further demonstrate that the unfolded protein response (UPR) is activated in the GRP170-deficient mouse. Notably, the UPR is also activated in AKI when originating from various other etiologies, including ischemia, sepsis, glomerulonephritis, nephrotic syndrome, and transplant rejection. Our work establishes the central role of GRP170 in kidney homeostasis and directly links molecular chaperone function to kidney injury.

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.

The mechanisms that link visceral mechanosensation to the perception of internal organ status (i.e., interoception) remain elusive. In response to bladder filling, the urothelium releases ATP, which is hypothesized to stimulate voiding function by communicating the degree of bladder fullness to subjacent tissues, including afferent nerve fibers. To determine if PIEZO channels function as mechanosensors in these events, we generated conditional urothelial Piezo1-, Piezo2-, and dual Piezo1/2-knockout (KO) mice. While functional PIEZO1 channels were expressed in all urothelial cell layers, Piezo1-KO mice had a limited phenotype. Piezo2 expression was limited to a small subset of superficial umbrella cells, yet male Piezo2-KO mice exhibited incontinence (i.e., leakage) when their voiding behavior was monitored during their active dark phase. Dual Piezo1/2-KO mice had the most affected phenotype, characterized by decreased urothelial responses to mechanical stimulation, diminished ATP release, bladder hypoactivity in anesthetized Piezo1/2-KO females but not males, and urinary incontinence in both male and female Piezo1/2-KO mice during their dark phase but not inactive light one. Our studies reveal that the urothelium functions in a sex- and circadian rhythm-dependent manner to link urothelial PIEZO1/2 channel-driven mechanotransduction to normal voiding function and behavior, and in the absence of these signals, bladder dysfunction ensues.

Expression and distribution of PIEZO1 in the mouse urinary tract.

The proper function of the organs that make up the urinary tract (kidneys, ureters, bladder, and urethra) depends on their ability to sense and respond to mechanical forces, including shear stress and wall tension. However, we have limited understanding of the mechanosensors that function in these organs and the tissue sites in which these molecules are expressed. Possible candidates include stretch-activated PIEZO channels (PIEZO1 and PIEZO2), which have been implicated in mechanically regulated body functions including touch sensation, proprioception, lung inflation, and blood pressure regulation. Using reporter mice expressing a COOH-terminal fusion of Piezo1 with the sequence for the tandem-dimer Tomato gene, we found that PIEZO1 is expressed in the kidneys, ureters, bladder, and urethra as well as organs in close proximity, including the prostate, seminal vesicles and ducts, ejaculatory ducts, and the vagina. We further found that PIEZO1 expression is not limited to one cell type; it is observed in the endothelial and parietal cells of the renal corpuscle, the basolateral surfaces of many of the epithelial cells that line the urinary tract, the interstitial cells of the bladder and ureters, and populations of smooth and striated muscle cells. We propose that in the urinary tract, PIEZO1 likely functions as a mechanosensor that triggers responses to wall tension.

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding.

The epithelial junctional complex, composed of tight junctions, adherens junctions, desmosomes, and an associated actomyosin cytoskeleton, forms the apical junctional ring (AJR), which must maintain its continuity in the face of external mechanical forces that accompany normal physiological functions. The AJR of umbrella cells, which line the luminal surface of the bladder, expands during bladder filling and contracts upon voiding; however, the mechanisms that drive these events are unknown. Using native umbrella cells as a model, we observed that the umbrella cell's AJR assumed a nonsarcomeric organization in which filamentous actin and ACTN4 formed unbroken continuous rings, while nonmuscle myosin II (NMMII) formed linear tracts along the actin ring. Expansion of the umbrella cell AJR required formin-dependent actin assembly, but was independent of NMMII ATPase function. AJR expansion also required membrane traffic, RAB13-dependent exocytosis, specifically, but not trafficking events regulated by RAB8A or RAB11A. In contrast, the voiding-induced contraction of the AJR depended on NMMII and actin dynamics, RHOA, and dynamin-dependent endocytosis. Taken together, our studies indicate that a mechanism by which the umbrella cells retain continuity during cyclical changes in volume is the expansion and contraction of their AJR, processes regulated by the actomyosin cytoskeleton and membrane trafficking events.

Age-related endolysosome dysfunction in the rat urothelium.

Lysosomal dysfunction is associated with a number of age-related pathologies that affect all organ systems. While much research has focused on neurodegenerative diseases and aging-induced changes in neurons, much less is known about the impact that aging has on lower urinary tract function. Our studies explored age-dependent changes in the content of endo-lysosomal organelles (i.e., multivesicular bodies, lysosomes, and the product of their fusion, endolysosomes) and age-induced effects on lysosomal degradation in the urothelium, the epithelial tissue that lines the inner surface of the bladder, ureters, and renal pelvis. When examined by transmission electron microscopy, the urothelium from young adult rats (~3 months), mature adult rats (~12 months), and aged rats (~26 months old) demonstrated a progressive age-related accumulation of aberrantly large endolysosomes (up to 7µm in diameter) that contained undigested content, likely indicating impaired degradation. Stereological analysis confirmed that aged endolysosomes occupied approximately 300% more volume than their younger counterparts while no age-related change was observed in multivesicular bodies or lysosomes. Consistent with diminished endolysosomal degradation, we observed that cathepsin B activity was significantly decreased in aged versus young urothelial cell lysates as well as in live cells. Further, the endolysosomal pH of aged urothelium was higher than that of young adult (pH 6.0 vs pH 4.6). Our results indicate that there is a progressive decline in urothelial endolysosomal function during aging. How this contributes to bladder dysfunction in the elderly is discussed.

RAB27B requirement for stretch-induced exocytosis in bladder umbrella cells.

Umbrella cells, which must maintain a tight barrier, modulate their apical surface area during bladder filling by exocytosis of an abundant, subapical pool of discoidal- and/or fusiform-shaped vesicles (DFVs). Despite the importance of this trafficking event for bladder function, the pathways that promote DFV exocytosis remain to be identified. We previously showed that DFV exocytosis depends in part on a RAB11A-RAB8A-MYO5B network, but RAB27B is also reported to be associated with DFVs, and knockout mice lacking RAB27B have fewer DFVs. However, the RAB27B requirements for DFV exocytosis and the relationship between RAB27B and the other umbrella cell-expressed RABs remains unclear. Using a whole bladder preparation, we observed that filling-induced exocytosis of human growth hormone-loaded DFVs was significantly inhibited when RAB27B expression was downregulated using shRNA. RAB27A was also expressed in rat urothelium; however, RAB27A-specific shRNAs did not inhibit exocytosis, and the combination of RAB27A and RAB27B shRNAs did not significantly affect DFV exocytosis more than treatment with RAB27B shRNA alone. RAB27B and RAB11A showed a small degree of overlap when quantified using Squassh segmentation software, and expression of dominant-active or dominant-negative mutants of RAB11A or RAB8A, or expression of a RAB11A-specific shRNA, had no significant effect on the size, number, or intensity of RAB27B-positive DFVs. Likewise, treatment with RAB27B-specific shRNA had no effect on RAB11A-positive DFV parameters. We conclude that RAB27B, but not RAB27A, regulates DFV exocytosis in bladder umbrella cells in a manner that may be parallel to the previously described RAB11A-RAB8A-MYO5B pathway.

Urothelial proliferation and regeneration after spinal cord injury.

The basal, intermediate, and superficial cell layers of the urothelium undergo rapid and complete recovery following acute injury; however, the effects of chronic injury on urothelial regeneration have not been well defined. To address this discrepancy, we employed a mouse model to explore urothelial changes in response to spinal cord injury (SCI), a condition characterized by life-long bladder dysfunction. One day post SCI there was a focal loss of umbrella cells, which are large cells that populate the superficial cell layer and normally express uroplakins (UPKs) and KRT20, but not KRT5, KRT14, or TP63. In response to SCI, regions of urothelium devoid of umbrella cells were replaced with small superficial cells that lacked KRT20 expression and appeared to be derived in part from the underlying intermediate cell layer, including cells positive for KRT5 and TP63. We also observed KRT14-positive basal cells that extended thin cytoplasmic extensions, which terminated in the bladder lumen. Both KRT14-positive and KRT14-negative urothelial cells proliferated 1 day post SCI, and by 7 days, cells in the underlying lamina propria, detrusor, and adventitia were also dividing. At 28 days post SCI, the urothelium appeared morphologically patent, and the number of proliferative cells decreased to baseline levels; however, patches of small superficial cells were detected that coexpressed UPKs, KRT5, KRT14, and TP63, but failed to express KRT20. Thus, unlike the rapid and complete restoration of the urothelium that occurs in response to acute injuries, regions of incompletely differentiated urothelium were observed even 28 days post SCI.

Increased urothelial paracellular transport promotes cystitis.

Changes in the urothelial barrier are observed in patients with cystitis, but whether this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 vs. controls transduced with green fluorescent protein. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.

TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the guanine nucleotide-exchange factors and GTPase-activating proteins (GAPs) that regulate its GTP-GDP cycle. We observed that in the presence of Mg(2+) (2.5 mM), TBC1D9B interacted via its Tre2-Bub2-Cdc16 (TBC) domain with Rab11a, Rab11b, and Rab4a in a nucleotide-dependent manner. However, only Rab11a was a substrate for TBC1D9B-stimulated GTP hydrolysis. At limiting Mg(2+) concentrations (<0.5 mM), Rab8a was an additional substrate for this GAP. In polarized Madin-Darby canine kidney cells, endogenous TBC1D9B colocalized with Rab11a-positive recycling endosomes but less so with EEA1-positive early endosomes, transferrin-positive recycling endosomes, or late endosomes. Overexpression of TBC1D9B, but not an inactive mutant, decreased the rate of basolateral-to-apical IgA transcytosis--a Rab11a-dependent pathway--and shRNA-mediated depletion of TBC1D9B increased the rate of this process. In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor. Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A. We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

Bladder filling and voiding affect umbrella cell tight junction organization and function.

Epithelial cells are continuously exposed to mechanical forces including shear stress and stretch, although the effect these forces have on tight junction (TJ) organization and function are poorly understood. Umbrella cells form the outermost layer of the stratified uroepithelium and undergo large cell shape and surface area changes during the bladder cycle. Here we investigated the effects of bladder filling and voiding on the umbrella cell TJ. We found that bladder filling promoted a significant increase in the length of the TJ ring, which was quickly reversed within 5 min of voiding. Interestingly, when isolated uroepithelial tissue was mounted in Ussing chambers and exposed to physiological stretch, we observed a 10-fold drop in both transepithelial electrical resistance (TER) and the umbrella cell junctional resistance. The effects of stretch on TER were reversible and dependent on the applied force. Furthermore, the integrity of the umbrella cell TJ was maintained in the stretched uroepithelium, as suggested by the limited permeability of biotin, fluorescein, and ruthenium red. Finally, we found that depletion of extracellular Ca(2+) by EGTA completely disrupted the TER of unstretched, but not of stretched uroepithelium. Taken together, our studies indicate that the umbrella cell TJ undergoes major structural and functional reorganization during the bladder cycle. The impact of these changes on bladder function is discussed.

A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells.

Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a-Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.