Wily G. Ruiz

ATP and purinergic receptor–dependent membrane traffic in bladder umbrella cells

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

Primary Uroepithelial Cultures A MODEL SYSTEM TO ANALYZE UMBRELLA CELL BARRIER FUNCTION

Despite almost 25 years of effort, the development of a highly differentiated and functionally equivalent cell culture model of uroepithelial cells has eluded investigators. We have developed a primary cell culture model of rabbit uroepithelium that consists of an underlying cell layer that interacts with a collagen substratum, an intermediate cell layer, and an upper cell layer of large (25–100 μm) superficial cells. When examined at the ultrastructural level, the superficial cells formed junctional complexes and had an asymmetric unit membrane, a hallmark of terminal differentiation in bladder umbrella cells. These cultured “umbrella” cells expressed uroplakins and a 27-kDa uroepithelial specific antigen that assembled into detergent-resistant asymmetric unit membrane particles. The cultures had low diffusive permeabilities for water (2.8 × 10−4 cm/s) and urea (3.0 × 10−7 cm/s) and high transepithelial resistance (>8000 Ω cm2) was achieved when 1 mmCaCl2 was included in the culture medium. The cell cultures expressed an amiloride-sensitive sodium transport pathway and increases in apical membrane capacitance were observed when the cultures were osmotically stretched. The described primary rabbit cell culture model mimics many of the characteristics of uroepithelium found in vivo and should serve as a useful tool to explore normal uroepithelial function as well as dysfunction as a result of disease.

Both Microtubules and Actin Filaments Are Required for Efficient Postendocytotic Traffic of the Polymeric Immunoglobulin Receptor in Polarized Madin-Darby Canine Kidney Cells

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.

Compensatory endocytosis in bladder umbrella cells occurs through an integrin‐regulated and RhoA‐ and dynamin‐dependent pathway

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin‐independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding‐stimulated CE, which depended on β1 integrin‐associated signalling pathways, occurred by a dynamin‐, actin‐, and RhoA‐regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO‐1‐positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin‐independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA‐ and dynamin‐dependent pathway, and terminates in degradation and not recapture of membrane in DFV.

Rab11a-dependent exocytosis of discoidal/fusiform vesicles in bladder umbrella cells

The discoidal/fusiform vesicles (DFV) of bladder umbrella cells undergo regulated exocytosis in response to stretch, but little is known about their biogenesis or the molecular machinery that modulates this process. We observed that Rab11a was expressed in umbrella cells (but not Rab11b or Rab25) and was associated with DFV. Using adenovirus-mediated delivery we transduced umbrella cells in situ with either dominant active (DA) or dominant negative (DN) mutants of Rab11a. DA-Rab11a stimulated an increase in apical surface area in the absence of stretch, whereas DN-Rab11a inhibited stretch-induced changes. Endocytosed fluid and membrane markers had little access to Rab11a-positive DFV, but virally expressed human growth hormone (hGH), a secretory protein, was packaged into DFV. Whereas expression of DA-Rab11a stimulated release of hGH into the bladder lumen, expression of DN-Rab11a had the opposite effect. Our results indicate that DFV may be biosynthetic in nature and that their exocytosis depends on the activity of the Rab11a GTPase.

Disruption of guinea pig urinary bladder permeability barrier in noninfectious cystitis

Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells.

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein‐tagged wild‐type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin‐Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant‐negative RhoBN19 or dominant‐active RhoBV14 altered postendocytic traffic of ligand‐receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral‐to‐apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.

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.

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 GEFs and GAPs that regulate its GTP-GDP cycle. TBC1D9B is identified as a Rab11a GAP in MDCK cells, where it regulates the Rab11a-dependent basolateral-to-apical transcytotic pathway.

Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+–ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.