Urea transport in MDCK cells that are stably transfected with UT-A1

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 ·cm². Transepithelial [¹⁴C]urea fluxes were measured at 37°C in a HCO₃^–/CO₂ buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [¹⁴C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells     

Mature N-linked glycans facilitate UT-A1 urea transporter lipid raft compartmentalization

The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature N-acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains.     

Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis

Urea movement across plasmamembranes is modulated by specialized transporter proteins that areproducts of two genes, termed UT-A and UT-B. These proteins play keyroles in the urinary concentrating mechanism and fluid homeostasis. Wehave isolated and characterized a 1.4-kb cDNA from testes encoding anew isoform (UT-A5) belonging to the UT-A transporter family. Forcomparison, we also isolated a 2.0-kb cDNA from mouse kidneyinner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNAhas a putative open reading frame encoding a 323-amino acidprotein, making UT-A5 the smallest UT-A family member in terms ofmolecular size. Its putative topology is of particular interest,because it calls into question earlier models of UT-A transporterstructure. Expression of UT-A5 cRNA in Xenopus oocytesmediates phloretin-inhibitable urea uptake and does not translocatewater. The distribution of UT-A5 mRNA is restricted to the peritubularmyoid cells forming the outermost layer of the seminiferous tubuleswithin the testes and is not detected in kidney. UT-A5 mRNA levels arecoordinated with the stage of testes development and increase 15 dayspostpartum, commensurate with the start of seminiferous tubule fluid movement.

     

Loss of N-linked glycosylation reduces urea transporter UT-A1 response to vasopressin

The vasopressin-regulated urea transporter (UT)-A1 is a transmembrane protein with two glycosylated forms of 97 and 117 kDa; both are derived from a single 88-kDa core protein. However, the precise molecular sites and the function for UT-A1 N-glycosylation are not known. In this study, we compared Madin-Darby canine kidney cells stably expressing wild-type (WT) UT-A1 to Madin-Darby canine kidney cell lines stably expressing mutant UT-A1 lacking one (A1m1, A1m2) or both glycosylation sites (m1m2). Site-directed mutagenesis revealed that UT-A1 has two glycosylation sites at Asn-279 and -742. Urea flux is stimulated by 10 nM vasopressin (AVP) or 10 microM forskolin (FSK) in WT cells. In contrast, m1m2 cells have a delayed and significantly reduced maximal urea flux. A 15-min treatment with AVP and FSK significantly increased UT-A1 cell surface expression in WT but not in m1m2 cells, as measured by biotinylation. We confirmed this finding using immunostaining. Membrane fractionation of the plasma membrane, Golgi, and endoplasmic reticulum revealed that AVP or FSK treatment increases UT-A1 abundance in both Golgi and plasma membrane compartments in WT but not in m1m2 cells. Pulse-chase experiments showed that UT-A1 half-life is reduced in m1m2 cells compared with WT cells. Our results suggest that mutation of the N-linked glycosylation sites reduces urea flux by reducing UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. In vivo, inner medullary collecting duct cells may regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.     

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

Madin-Darby canine kidney (MDCK)I and Fisher rat thyroid (FRT) cells exhibit transepithelial electricalresistance (TER) values in excess of 5,000  · cm². When these cells wereincubated in the presence of various inhibitors of sphingolipidbiosynthesis, a >5-fold reduction of TER was observed without changesin the gate function for uncharged solutes or the fence function forapically applied fluorescent lipids. The localization of ZO-1 andoccludin was not altered between control and inhibitor-treated cells,indicating that the tight junction was still intact. Furthermore, thecomplexity of tight junction strands, analyzed by freeze-fracturemicroscopy, was not reduced. Once the inhibitor was removed and thecells were allowed to synthesize sphingolipids, a gradual recovery ofthe TER was observed. Interestingly, these inhibitors did not attenuatethe TER of MDCK II cells, a cell line that typically exhibits valuesbelow 800  · cm^2. These resultssuggest that glycosphingolipids play a role in regulating theelectrical properties of epithelial cells.

     

The UT-A1 urea transporter interacts with snapin, a SNARE-associated protein

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.     

Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking

The cytoskeleton participates in many aspects of transporter protein regulation. In this study, by using yeast two-hybrid screening, we identified the cytoskeletal protein actin as a binding partner with the UT-A1 urea transporter. This suggests that actin plays a role in regulating UT-A1 activity. Actin specifically binds to the carboxyl terminus of UT-A1. A serial mutation study shows that actin binding to UT-A1's carboxyl terminus was abolished when serine 918 was mutated to alanine. In polarized UT-A1-MDCK cells, cortical filamentous (F) actin colocalizes with UT-A1 at the apical membrane and the subapical cytoplasm. In the cell surface, both actin and UT-A1 are distributed in the lipid raft microdomains. Disruption of the F-actin cytoskeleton by latrunculin B resulted in UT-A1 accumulation in the cell membrane as measured by biotinylation. This effect was mainly due to inhibition of UT-A1 endocytosis in both clathrin and caveolin-mediated endocytic pathways. In contrast, actin depolymerization facilitated forskolin-stimulated UT-A1 trafficking to the cell surface. Functionally, depolymerization of actin by latrunculin B significantly increased UT-A1 urea transport activity in an oocyte expression system. Our study shows that cortical F-actin not only serves as a structural protein, but directly interacts with UT-A1 and plays an important role in controlling UT-A1 cell surface expression by affecting both endocytosis and trafficking, therefore regulating UT-A1 bioactivity.     

Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters

The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects. ubiquitin-proteasome pathway; urea transport; membrane localization     

Hyperosmotic NaCl and urea synergistically regulate the expression of the UT-A2 urea transporter in vitro and in vivo

The UT-A2 urea transporter is involved in the recycling of urea through the kidney, a process required to maintain high osmotic gradients. Dehydration increases UT-A2 expression in vivo. The tissue distribution of UT-A2 suggested that hyperosmolarity, and not vasopressin, might mediate this effect. We have analyzed the regulation of UT-A2 expression by ambiant osmolarity both in vitro (mIMCD3 cell line) and in vivo (rat kidney medulla). The UT-A2 mRNA was found to be synergistically up-regulated by a combination of NaCl and urea. Curiously, the UT-A2 protein was undetectable in this hypertonic culture condition, or after transfection of the UT-A2 cDNA, whereas it could be detected in HEK-293 transfected cells. Treating rats with furosemide, a diuretic which decreases the kidney interstitium osmolarity without affecting vasopressin levels, led to decreased levels of the UT-A2 protein. Our results show that the UT-A2 urea transporter is regulated by hyperosmolarity both in vitro and in vivo.     

Regulation of Nod1-mediated signaling pathways

Nod1 is a member of the NLR/Nod/CATERPILLER family. It acts as a sensor for intracellular bacteria by recognizing specific glycopeptides derived from peptidoglycan. Nod1 activation mediates distinct cellular responses including activation of MAP kinases, IL-8 release, apoptosis and suppression of several estrogen-dependent responses in MCF-7 cells. Here we have extended these studies by identifying key regulatory steps in Nod1-dependent signaling pathways. We provide multiple lines of data showing that Nod1-dependent apoptosis is a caspase 8-mediated event and that apoptosis requires RIP2. In contrast, several lines of evidence show that Nod1-dependent JNK activation and IL-8 production did not require the presence of caspase 8 but required activation of TAK1 as well as RIP2. Thus, we have identified several key control points that lie downstream of Nod1. This work provides the basis for further studies of the biological significance and regulation of the Nod1 pathway.     

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells

MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bre, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.     

Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity

In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Structure-activity analysis of thiourea analogs as inhibitors of UT-A and UT-B urea transporters

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC₅₀ of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC₅₀ of ~ 0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC₅₀ 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC₅₀ > 15 vs. 2.8 mM).     

Glycoforms of UT-A3 urea transporter with poly-N-acetyllactosamine glycosylation have enhanced transport activity

Urea transporters UT-A1 and UT-A3 are both expressed in the kidney inner medulla. However, the function of UT-A3 remains unclear. Here, we found that UT-A3, which comprises only the NH(2)-terminal half of UT-A1, has a higher urea transport activity than UT-A1 in the oocyte and that this difference was associated with differences in N-glycosylation. Heterologously expressed UT-A3 is fully glycosylated with two glycoforms of 65 and 45 kDa. By contrast, UT-A1 expressed in HEK293 cells and oocytes exhibits only a 97-kDa glycosylation form. We further found that N-glycans of UT-A3 contain a large amount of poly-N-acetyllactosamine. This highly glycosylated UT-A3 is more stable and is enriched in lipid raft domains on the cell membrane. Kifunensine, an inhibitor of α-mannosidase that inhibits N-glycan processing beyond high-mannose-type N-glycans, significantly reduced UT-A3 urea transport activity. We then examined the native UT-A1 and UT-A3 glycosylation states from kidney inner medulla and found the ratio of 65 to 45 kDa in UT-A3 is higher than that of 117 to 97 kDa in UT-A1. The highly stable expression of highly glycosylated UT-A3 on the cell membrane in kidney inner medulla suggests that UT-A3 may have an important function in urea reabsorption.