AJP - Renal Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 294: F518-F524, 2008. First published January 2, 2008; doi:10.1152/ajprenal.00349.2007
0363-6127/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/F518    most recent
00349.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fröhlich, O.
Right arrow Articles by Sands, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fröhlich, O.
Right arrow Articles by Sands, J. M.

Stimulation of UT-A1-mediated transepithelial urea flux in MDCK cells by lithium

Otto Fröhlich,1 Deepak Aggarwal,2 Janet D. Klein,2 Kimilia J. Kent,2 Yuan Yang,1 Robert B. Gunn,1,{dagger} and Jeff M. Sands1,2

1Department of Physiology, Emory University and 2Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia

Submitted 26 July 2007 ; accepted in final form 30 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Trans-epithelial tracer urea flux across Madin-Darby canine kidney (MDCK) cells permanently expressing the urea transporter UT-A1 is stimulated by agents that activate the cAMP signaling pathway, such as vasopressin or forskolin, thus mimicking the activation of urea permeability in the inner medullary collecting duct in the presence of vasopressin. Here, we report that UT-A1-mediated urea flux is also activated two-to-threefold over background by exposing the cells to media containing LiCl. This is in contrast to reports on cortical and medullary collecting duct tubules where acute and chronic exposure to lithium (Li) suppresses the osmotic water permeability, which is also regulated by cAMP levels. The Li concentration dependence of urea flux activation was linear up to 150 mM Li. Li activated only from the basolateral side where its effect was inhibited by amiloride, presumably because Li entered the cells through a basolateral Na-H exchanger. Li and IBMX, which also weakly activated urea flux, greatly augmented each others' stimulatory effect on urea flux. However, cellular cAMP levels did not rise commensurately with urea fluxes, and even though Li augments the activation by forskolin, it greatly inhibits the forskolin-induced formation of cAMP. These results suggest that the effect of Li in this MDCK model of renal cells does not involve cAMP or at least utilizes an additional signaling pathway independent of cAMP.

cyclic AMP; Madin-Darby canine kidney cells

LITHIUM TREATMENT HAS PROVED to be an effective pharmacological agent to control, among others, bipolar affective disorder (19), through mechanisms that are not yet well understood. Unfortunately, lithium (Li) treatment has toxic side effects on several organ systems including the kidney (31). Li treatment can result in an impaired ability to reabsorb water and therefore to concentrate the urine, leading in some cases to polyuria and polydipsia (1). Prolonged treatment can render this impairment progressively irreversible and nonresponsive to the action of vasopressin (AVP) (21, 25, 32).

Studies with animal models of Li treatment confirmed the polyuric effect of Li treatment (3, 35) and work with isolated renal tubular segments demonstrated that Li inhibits the AVP-induced water permeability, with the inhibition occurring at the level of cAMP generation (3–5). In rat medullary collecting duct cells, it has been suggested that this reduced cAMP generation results from activation of the inhibitory GTP binding protein, Gi (35), whereas in cultured LLC-PK1 cells, Li appears to inhibit adenylate cyclase activity by disrupting the interaction between adenylate cyclase and the stimulatory GTP binding protein, Gs (10).

More recent experiments with animals demonstrated that chronic Li treatment also leads to altered expression of transport proteins in the collecting duct. Of particular importance with respect to water handling is the observation that the AVP-regulated water channel aquaporin-2 (AQP-2) is dramatically downregulated in rats after 25 days of Li treatment (2, 15, 21). The same effect has been noted in a cultured cortical collecting duct cell line (20). Among the other aquaporins found in the kidney, AQP-3 is also downregulated, whereas AQP-1 and AQP-4 and several cation transporters are unaffected (17), but evidence is accumulating that chronic Li might have a fairly broad effect on gene expression (2, 28).

Much less attention has been given to the renal urea transporter whose physiological role is to maintain a high interstitial medullary urea concentration and to support the formation of the cortico-medullary osmotic gradient (29). Klein et al. (15) showed that both urea transporter isogenes, UT-A1 in the renal tubule and UT-B in the vasa recta, are downregulated after 25 days of Li feeding. Furthermore, Li treatment reduced the phosphorylation of UT-A1 in response to AVP. However, there is no report in the literature on a possible effect of acute Li exposure on urea transport activity in the renal collecting duct.

The purpose of the present experiments was to explore the effect of acute Li treatment on short-term regulation of urea transport in a cellular model. We developed a permanently transfected Madin-Darby canine kidney (MDCK) cell line that constitutively expresses the UT-A1 protein. In the absence of agents known to stimulate the cellular cAMP levels, these cells exhibit a low urea permeability, comparable to that expected for the permeability of the lipid bilayer, but in the presence of AVP or forskolin, the transepithelial urea permeability is greatly enhanced through activation of UT-A1 (7, 8). The goal of the experiments was to test whether Li interfered with the activation of urea transport by AVP or forskolin and to compare this with what had been observed for osmotic water permeability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. All chemicals were reagent grade. Forskolin and the protein kinase inhibitor H-89 (Sigma Chemicals, Sigma, St. Louis, MO) were dissolved in DMSO as a 1,000x stock solution. AVP (Sigma) was dissolved in water as a 10–4 M stock solution. The AVP stock solution was stored frozen at –70°C and thawed only once, further diluted for final use and the unused portion was discarded after the experiment.

UT-A1-expressing MDCK cells. The cell line used in these experiments, MDCK-UT-A1, is permanently transfected with the gene encoding the rat UT-A1 form of the urea transporter. The UT-A1 protein is constitutively expressed, but unless an activator such as AVP or forskolin is added, there is no significant UT-A1-mediated urea transport across the epithelium (8). The method of construction of this cell line and of characterizing the activation of urea transport by activators of cAMP levels has been described earlier (8). Cells were maintained in DMEM (GIBCO). To prepare them for a flux experiment, the cells were plated at a density of 2 x 105 cells/cm2 on collagen-coated Costar Transwell inserts (Corning, Acton, MA). Confluency was generally obtained after 4–5 days, at which point the filters reached a transepithelial resistance of typically >800 {Omega}·cm2.

Media. The flux media contained (in mM) 140 NaCl ("Na medium") or 140 LiCl ("Li medium"), 5 urea, 1.5 K2HPO4, 5.5 glucose, 0.8 MgCl2, 1.0 CaCl2, and 24 HEPES, titrated to pH 7.64 at room temperature. To vary the Li concentration, the desired proportions of Na medium and Li medium were mixed. In most experiments, the flux medium was the same on both sides of the epithelium.

Urea flux measurements. Details of the method have been described previously (7, 8). Apical-to-basolateral tracer urea flux at 37°C was measured with cells grown to confluence in Transwell inserts. To start an experiment, an insert was placed into a well of a 12-well culture dish that resided on a thermostatted block, the (apical) medium was removed and from the insert replaced with 14C-urea-containing medium. At 3-min intervals, the insert was moved into a new well containing fresh flux medium. The flux was calculated from the radioactivity that had accumulated in the well (basolateral solution) during this period. The experimental setup permitted eight inserts to be fluxed in parallel in one experimental series. Typically, a flux experiment consisted of determining the unstimulated flux over three time points (9 min), followed by an experimental period of 54 min and ending with three time points during which the inserts were placed in medium containing 100 mM dimethylurea (DMU). The urea transport inhibitor DMU served to demonstrate that the activated flux was mediated by the urea transporter and not due to a nonspecific leak pathway. The summary bar graphs in the figures are the averages and standard deviations of the last three to four time points of the activation period (typically 45–54 min after the start of flux activation).

Cellular cAMP determinations. Cells grown on Transwell filter inserts and exposed to the desired experimental conditions were harvested by transferring the filter inserts into an empty well of a culture dish and removing the apical medium. When used after the completion of a flux experiment, the cells were quickly washed with flux medium to remove the 14C-urea before further handling. To denature the cells and inhibit degradation of cellular cAMP, 400 µl 0.1 M HCl were added to the insert. Cells were scraped off the filter, collected together with the HCl supernatant in a microcentrifuge tube, and kept at room temperature for 10–15 min. They were stored in a –80°C freezer until the time they were analyzed. At that time, the samples were thawed at room temperature and centrifuged to pellet all insoluble cellular material. The cAMP assay was performed on typically 50 µl of the supernatant using the Direct Cyclic AMP immunoassay kit from Assay Designs (Ann Arbor, MI) and following the manual's instructions. Each filter sample was assayed in duplicate. We added no phosphodiesterase inhibitor such as IBMX to the cells, unless specifically indicated for a given experiment.

Statistical analysis. Most figures report the results of a set of flux experiments on eight Transwell inserts. For the summary histograms, averages and standard deviations were calculated from the last four time points of the experimental treatment period (before the urea transport inhibitor DMU was added to test for UT-specific fluxes). Statistical significance is indicated by an asterisk above the bar. The summary graphs of Fig. 8 are the result of five separate series of experiments in which cells were both subjected to flux measurements and then harvested to determine cAMP levels. Statistical significance was determined by paired t-test.


Figure 8
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 8. Combined measurements of urea fluxes and cellular cAMP contents. Data are averages and standard deviations of 5 separate experimental series. At the end of the flux experiment (A), the cells from each filter were collected for determination of cAMP contents (B). Paired comparison of the measurements in Na vs. Li medium were performed to determine statistically significant differences (indicated by *) between the 2 groups.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute exposure to Li enhances AVP-induced urea transport. In experiments reported in the literature on isolated renal tubules, acute exposure to Li-containing medium attenuated the stimulatory effect of AVP on osmotic water permeability (4, 5). We therefore tested the effect of Li exposure on the activation of urea transport by AVP in our MDCK-UT-A1 cells (Fig. 1). In agreement with previous experiments, in the absence of an activator the trans-epithelial urea flux in the presence of 5 mM urea was low (1–3 nmol·cm–2·min–1) and corresponded to the urea permeability of the plasma membrane (8). When the cells were exposed to the vasopressin analog AVP, urea flux rose over the next 30 min to typically 10–15 nmol·cm–2·min–1. In contrast, when the cells were transferred into a medium in which LiCl replaced NaCl as major salt, we observed two effects of Li. First, in the presence of Li alone, urea flux gradually increased to typically 5 nmol·cm–2·min–1 over 60 min. Second, the activation of urea flux by AVP was greatly enhanced, with fluxes rising to typically 40 nmol·cm–2·min–1.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 1. Time course of trans-epithelial urea flux activation by vasopressin in Na+- or lithium (Li+)-containing medium. In all experiments, the apical side contained Na medium and the composition of the basolateral medium was varied. A: {triangleup} and bullet, no AVP added; {lozenge} and {blacksquare}, 10–8 M AVP added basolaterally. Open symbols, Na medium; filled symbols, Li medium. Li medium replaced Na medium starting with the fourth time point. AVP (10–8 M) was present on the basolateral side during the period indicated by "treatment." During the last 3 time points, 100 mM dimethylurea (DMU) was added to the basolateral medium. B: summary graph of the data of A, obtained by averaging the last 4 time point samples before the addition of DMU. Li significantly enhances urea fluxes both in the absence and presence of AVP.

 
Acute exposure to Li enhances the effect of other activators of urea flux. To test whether this effect was specific to AVP or could also be observed with other agents known to increase intracellular cAMP levels, we measured urea fluxes in cells in the presence of 10 µM forskolin, 200 µM phosphodiesterase inhibitor IBMX, and a combination of 200 mM IBMX and 10–8 M AVP (Fig. 2). As found with AVP, the effect of all three treatments was also significantly augmented by the presence of Li in the medium. Even the effect of forskolin, the strongest single activator in our previous experiments (7, 8), was further enhanced in Li medium.


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Summary graphs of the effect of Li on the activation of urea transport by known activators of urea flux. The concentrations used were 200 µM IBMX, 10 µM forskolin (FSK), and 10–8 M AVP. In all 4 conditions, flux was significantly stimulated by the presence of Li compared with Na.

 
Li enters the cell from the basolateral side on a Na-H exchanger. To test the concentration dependence of the Li effect, we mixed NaCl and LiCl media at several different ratios. Furthermore, to enhance the effect of Li, which by itself exhibited only moderate activation, we added IBMX (200 µM) to the basolateral medium. Figure 3 shows that the Li concentration dependence was essentially linear in the range up to at least 150 mM.


Figure 3
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 3. Concentration dependence of the Li effect. Basolateral Li concentration was varied by mixing Na and Li media in the ratios indicated. The data suggest an approximately linear increase with Li concentration. IBMX (200 µM) was present in all experiments to enhance the Li effect.

 
Figure 4 shows that the Li effect was asymmetric. When Li was present only on the apical side, it had no activating effect on urea transport. On the other hand, when added to the basolateral side only, its effect was comparable to that seen with Li present on both sides. However, we were able to elicit an effect by apical Li when we added the ionophore monensin to the apical side in the presence of Li (Fig. 4). This suggests that the Li effect is caused by Li entering the cell and not simply by binding to an extracellular basolateral site.


Figure 4
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 4. Sidedness of Li effect. Li medium was present only on one side, basolateral or apical. Li alone had no stimulatory effect from the apical side. Flux activation by apical Li was only observed in the presence of the cation ionophore monensin (100 µM added apically).

 
Furthermore, the Li effect appears to be specific to Li over other cations. In media in which NaCl was replaced isotonically by choline-Cl or MgCl2, urea transport remained constant and at the same level as found for NaCl medium (Fig. 5).


Figure 5
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 5. Urea fluxes in the presence of other cations substituted isoosmotically for Na. Only Li exerted a stimulator effect.

 
To determine how Li might enter the cell, we employed several inhibitors of transport systems that are known to carry Li as a possible substrate. Figure 6 shows that neither chlorothiazide, an inhibitor of Na-Cl cotransport, nor furosemide, an inhibitor of Na-K-Cl cotransport, had any noticeable effect on the way Li activated urea transport. Neither was there an effect of the Na pump inhibitor ouabain (Fig. 6B). However, amiloride and its derivatives, especially those with a higher affinity for the Na-H exchanger, significantly suppressed the effect of Li. It therefore appears that Li entered the cells by means of a basolateral Na-H exchanger.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 6. Effect of cation transport inhibitors on Li-stimulated urea fluxes. A: chlorothiazide: Na-Cl– cotransport; furosemide: Na-K-Cl– cotransport; amiloride: Na-H exchanger (low affinity) and epithelial Na channel (high affinity). B: ouabain: Na-K-ATPase; benzamil and hexamethylene amiloride (HMA) are higher-affinity inhibitors of Na-H exchange than amiloride.

 
Li effect does not involve protein kinase A. The generally held notion of how AVP activates urea transport is by binding to the V2 receptor which through the Gs protein activates adenylate cyclase and raises cAMP levels (13), in turn activating protein kinase A (PKA). In a previous study, we suggested that phosphorylation of the urea transporter and/or another mediator protein leads to an increased trans-epithelial urea permeability (7, 37). We also showed that the PKA inhibitor, H-89, blocked approximately one-half of the urea transport activation by AVP or forskolin in MDCK-UT-A1 cells, which suggests that at least some of the activation of was mediated by PKA (7). However, when we tested H-89 for its effect on the activation of urea transport by Li, we found that it had no inhibitory effect (Fig. 7). If anything, according to the data in Fig. 7, at high concentrations H-89 actually appeared to enhance the effect of Li.


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 7. Effect of the protein kinase A inhibitor, H89, on Li-stimulated urea fluxes. Basolateral H89 was present throughout the entire experimental time course, including the first 3 time points before the switch to Li medium.

 
Li can activate urea transport without stimulating cellular cAMP formation. Since activation of urea transport has, in general, been associated with increased cellular cAMP levels, we determined to which extent Li affected the cAMP levels in the MDCK-UT-A1 cells. In five series of experiments we combined measurements of urea flux after exposure to AVP, forskolin, and IBMX, in control (NaCl) media and in LiCl media, with measurements of the cellular cAMP content at the end of the flux experiment. In agreement with the ealier experiments, Li enhanced the activation of urea flux in the absence and presence of flux activators AVP, forskolin, and IBMX (Fig. 8A). In particular, the combined stimulatory effect of Li and IBMX was significantly stronger than expected from the individual effects of Li and IBMX on the urea permeability. However, cAMP levels did not vary in accordance with the urea fluxes. Li by itself slightly stimulated cAMP levels but had no significant effect on cAMP levels in the presence of AVP and IBMX. However, it significantly suppressed forskolin-dependent cAMP formation (Fig. 8B).

To test whether the observed Li-dependent urea flux activation was due to increased movement of UT-A1 protein into the apical membrane, we also performed biotinylation experiments. Previously, we demonstrated that exposing UT-A1-MDCK cells to dDAVP or forskolin increased the amount UT-A1 protein in the apical membrane (14). However, exposing UT-A1-MDCK cells to Li under the same conditions as the flux experiments had no influence on the amount of apical biotinylation of UT-A1 protein (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we tested our MDCK-UT-A1 cell line for its usefulness as a cellular model to investigate the acute effects of Li on the activation of inner medullary urea transport. The initial working hypothesis was that, since regulation of inner medullary urea permeability by AVP roughly parallels that of water permeability (33), Li would have similar effects on urea and water transport.

However, in apparent contrast to the generally found inhibitory effect of Li on osmotic water permeability, we found that, in the MDCK-UT-A1 cells, exposure to LiCl medium stimulated rather than inhibited the trans-epithelial permeability to tracer urea. This increased permeability was mediated specifically by the urea transporter protein, UT-A1, as evident from the return of the stimulated flux to baseline levels when the transport inhibitor DMU was added to the medium.

The Li effect was not duplicated by replacing NaCl with MgCl2 or choline-Cl instead of LiCl. Thus, the activation of urea transport was not caused by the absence of Na but by the presence of Li in the medium. Even though Li by itself was active only when present on the basolateral side, it did activate urea fluxes from the apical side when the ionophore monensin was present on the apical side at the same time. In addition, we observed that amiloride and its analogs were able to inhibit the effect of basolateral Li. Taken together, these results suggest that Li had to enter the cell to exert its activating effect and that it did so by being transported on a basolateral Na-H exchanger.

The apical/basolateral asymmetry of the Li effect observed is opposite to the asymmetry found in rabbit cortical collecting tubules where only luminal Li inhibited cAMP formation (4). The reason for this apparent discrepancy is not known but could lie in the different segmental origins of the two cell types, as MDCK cells exhibit properties reminiscent of medullary collecting duct cells. It is possible that cortical and medullary collecting ducts possess different Li entry pathways. For example, the rat cortical collecting duct is known to contain apical ENaC channels that are permeable to Li (9). Since amiloride has an inhibitory potency for ENaC channels several orders of magnitude higher than that apparent in our studies (near 1 mM; Fig. 6B), it is most likely that in the case of the MDCK cells Li entered through a basolateral Na-H exchanger. In support of this notion, MDCK cells have been demonstrated to possess a Na-H exchanger (27). It is interesting, though, that in the isolated rabbit cortical collecting tubule Li exerted its (inhibitory) effect on osmotic water permeability from the apical but not from the basolateral side (4). Perhaps in this case Li entered through apical Na channels. It is not clear from which side Li entered in the more recent study on mouse cortical collecting duct-derived MpkCCD cells (27) as Li was added to both sides of the cultured cells.

The Li concentrations used in most of our experiments were higher than those used in previous experiments studying the effect of acute Li exposure (150 vs. 10–20 mM) and significantly higher than the estimated plasma Li concentrations in chronically exposed animals (as low as 1 mM). Using such a high concentration was a useful tool to increase the experimental signal, but the apparently linear dependence of the stimulatory effect on the Li concentration (Fig. 3) suggests that activation of urea transport already occurs at less than 50 mM Li without an additional activator. It should be noted that in our experiments activation of urea transport could be observed as early as after 15 min of exposure, whereas in the published studies the tissues were exposed for up to several hours before being assayed.

A major difference between the present experiments and most studies reported in the literature is the mode of exposure to Li. As expected for a drug that is used as chronic treatment, most studies have explored the effect of long-term (many days to weeks) exposure on renal function. It is clear from these reports that Li exposure for several days or weeks not only affects the gene expression of AQP-2 (17, 21) and UT-A (15) but also that of other transporters (17, 18, 28) and enzymes (16, 26) and eventually even alters the cellular composition of the collecting duct (2). In the case of AQP-2 it had appeared likely that cAMP influenced gene expression through cAMP-responsive transcription elements (22, 36), but more recent experiments point toward a cAMP-independent effect of chronic Li treatment (20). Considering the broad effect of Li on a long list of enzymes (11), other signaling pathways could also be involved in this pleiotropic response to chronic Li exposure (7, 20). With respect to the present study, it would appear reasonable to presume that only a small number of signaling pathways, excluding transcriptional processes, is responsible for the observed acute upregulation of the urea permeability in MDCK-UT-A1 cells.

Li alone stimulated urea transport, and it also enhanced the stimulation by AVP, forskolin, and IBMX, activators known to raise intracellular cAMP levels. Even at a saturating concentration of forskolin urea flux was further stimulated by Li. The Li effect thus appears to be additive to that of the other agents, as if Li raised the cAMP levels even further. However, this would contradict previous suggestions that Li inhibits adenylate cyclase activity (3–5, 10, 35). Our cAMP results are consistent with Li inhibiting adenylyl cyclase because Li exposure greatly suppressed cAMP formation in the presence of forskolin. Alternatively, Li may also activate, directly or indirectly, a phosphodiesterase activity that is responsible for degrading cAMP. However, by itself Li did appear to raise the cAMP levels slightly. The mechanism for this weak stimulation is not clear. We can eliminate the involvement of PKA as a major participant in the activation process because H-89 had no effect on this activation. However, this observation by itself would not eliminate a more indirect activation pathway involving cAMP since we found that AVP- and forskolin-activated urea fluxes are only partially inhibited by H-89 (7). It is known that cAMP can activate the MAP kinase Erk1/2 cascade via Epac/cAMP-GEF proteins, guanine nucleotide exchange factors for the small GTPase Rap1 (6) which feeds into this signaling cascade. It will be important to examine, in the future, whether in the UT-A1-MDCK cells, this pathway is activated in parallel to urea flux activation by AVP or forskolin.

From the comparison of the Li effects on urea flux activation and on cAMP generation, it would appear that urea transport can be activated without a concurrent stimulation of cAMP formation. It is unknown through which alternative signaling pathway Li may affect urea permeability. The hydrated Li ion is similar in size to magnesium and is known to interfere with Mg-requiring enzymes, including several different phosphatases and kinases (11). However, the cAMP values measured in cell extracts do not necessarily reflect the cAMP concentrations relevant for activating urea fluxes. Recent studies on several cellular systems demonstrated that signaling pathways involving cAMP can be physically isolated from the bulk cytoplasmic phase. For example, pathway components (adenylyl cyclase, PKA, and/or phosphodiesterase) can be anchored to compartments that are diffusionally separated from the rest of the cell (12, 23, 30, 34).

Since one of the physiological end results of AVP binding to inner medullary collecting duct cells is activation of trans-epithelial urea transport, any signaling pathways to that effect necessarily converge at this point. Current evidence suggests that the point of convergence involves protein phosphorylation and trafficking of the UT-A1 urea transporter protein to the plasma membrane, similar to what has been found for the AQP-2 protein (14, 24). Therefore, one could surmise that the Li-mediated rise in trans-epithelial urea flux might also be accompanied by a UT-A1 protein shift into the apical membrane. However, we saw no such increase in the extent of biotinylation of UT-A1 protein upon exposure to Li (data not shown). This would suggest that activation of urea transport across UT-A1-MDCK cells involves not only trafficking to the membrane, but also modulation of individual channel activity after incorporation into the apical membrane.

In summary, the question about the detailed mechanism underlying the acute regulation of urea transport by Li is not clear. Cytoplasmic Li clearly has a suppressing effect on bulk cytoplasmic cAMP levels when these were raised by one of the traditional agonists, but it is possible that the cAMP that determines urea flux activity differs from the bulk cAMP because of diffusional isolation in a domain near the membrane. In addition, it will be of interest to examine the Li sensitivity of the components of the signaling cascade that governs the activation of urea transporter.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant PO1-DK-6152.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Froehlich, Emory Univ. School of Medicine, Dept. of Physiology, 605R Whitehead Bldg., 615 Michael St., Atlanta, GA 30322 (e-mail: otto.froehlich{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased June 25, 2005. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Boton R, Gaviria M, Batlle DC. Prevalence, pathogenesis, and treatment of renal dysfunction associated with chronic lithium therapy. Am J Kidney Dis 10: 329–345, 1987.[Web of Science][Medline]
  2. Christensen BM, Marples D, Kim YH, Wang W, Frokiær J, Nielsen S. Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI. Am J Physiol Cell Physiol 286: C952–C964, 2004.[Abstract/Free Full Text]
  3. Christensen S, Kusano E, Yusufi AN, Murayama N, Dousa TP. Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats. J Clin Invest 75: 1869–1879, 1985.[Web of Science][Medline]
  4. Cogan E, Abramow M. Inhibition by lithium of the hydroosmotic action of vasopressin in the isolated perfused cortical collecting tubule of the rabbit. J Clin Invest 77: 1507–1514, 1986.[Medline]
  5. Cogan E, Svoboda M, Abramow M. Mechanisms of lithium-vasopressin interaction in rabbit cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 252: F1080–F1087, 1987.[Abstract/Free Full Text]
  6. De Rooij J, Rehmann H, van Triest M, Cool RH, Wittinghofer A, Bos JL. Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J Biol Chem 275: 20829–20836, 2000.[Abstract/Free Full Text]
  7. Fröhlich O, Klein JD, Smith PM, Sands JM, Gunn RB. Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells. Am J Physiol Cell Physiol 291: C600–C606, 2006.[Abstract/Free Full Text]
  8. Fröhlich O, Klein JD, Smith PM, Sands JM, Gunn RB. Urea transport in MDCK cells that are stably transfected with UT-A1. Am J Physiol Cell Physiol 286: C1264–C1270, 2004.[Abstract/Free Full Text]
  9. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77: 359–396, 1997.[Abstract/Free Full Text]
  10. Goldberg H, Clayman P, Skorecki K. Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 255: F995–F1002, 1988.[Abstract/Free Full Text]
  11. Gould TD, Quiroz JA, Singh J, Zarate CA, Manji HK. Emerging experimental therapeutics for bipolar disorder: insights from the molecular and cellular actions of current mood stabilizers. Mol Psychiatry 9: 734–755, 2004.[CrossRef][Web of Science][Medline]
  12. Henn V, Stefan E, Baillie GS, Houslay MD, Rosenthal W, Klussmann E. Compartmentalized cAMP signalling regulates vasopressin-mediated water reabsorption by controlling aquaporin-2. Biochem Soc Trans 33: 1316–1318, 2005.[CrossRef][Medline]
  13. Ishikawa S. Cellular actions of arginine vasopressin in the kidney. Endocr J 40: 373–386, 1993.[Web of Science][Medline]
  14. Klein JD, Fröhlich O, Blount MA, Martin CF, Smith TD, Sands JM. Vasopressin increases plasma membrane accumulation of urea transporter UT-A1 in rat inner medullary collecting ducts. J Am Soc Nephrol 17: 2680–2686, 2006.[Abstract/Free Full Text]
  15. Klein JD, Gunn RB, Roberts BR, Sands JM. Downregulation of urea transporters in the renal inner medulla of lithium-fed rats. Kidney Int 61: 995–1002, 2002.[CrossRef][Web of Science][Medline]
  16. Kotnik P, Nielsen J, Kwon TH, Krzisnik C, Frokiær J, Nielsen S. Altered expression of COX-1, COX-2, and mPGES in rats with nephrogenic and central diabetes insipidus. Am J Physiol Renal Physiol 288: F1053–F1068, 2005.[Abstract/Free Full Text]
  17. Kwon TH, Laursen UH, Marples D, Maunsbach AB, Knepper MA, Frokiær J, Nielsen S. Altered expression of renal AQPs and Na+ transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 279: F552–F564, 2000.[Abstract/Free Full Text]
  18. Laursen UH, Pihakaski-Maunsbach K, Kwon TH, Ostergaard Jensen E, Nielsen S, Maunsbach AB. Changes of rat kidney AQP2 and Na, K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus. Nephron Exp Nephrol 97: e1–e16, 2004.[CrossRef][Medline]
  19. Lenox RH, McNamara RK, Papke RL, Manji HK. Neurobiology of lithium: an update. J Clin Psychiatry 59, Suppl 6: 37–47, 1998.
  20. Li Y, Shaw S, Kamsteeg EJ, Vandewalle A, Deen PM. Development of lithium-induced nephrogenic diabetes insipidus is dissociated from adenylyl cyclase activity. J Am Soc Nephrol 17: 1063–1072, 2006.[Abstract/Free Full Text]
  21. Marples D, Christensen S, Christensen EI, Ottosen PD, Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 95: 1838–1845, 1995.[Web of Science][Medline]
  22. Matsumura Y, Uchida S, Rai T, Sasaki S, Marumo F. Transcriptional regulation of aquaporin-2 water channel gene by cAMP. J Am Soc Nephrol 8: 861–867, 1997.[Abstract]
  23. McSorley T, Stefan E, Henn V, Wiesner B, Baillie GS, Houslay MD, Rosenthal W, Klussmann E. Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells. Eur J Cell Biol 85: 673–678, 2006.[CrossRef][Medline]
  24. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 92: 1013–1017, 1995.[Abstract/Free Full Text]
  25. Presne C, Fakhouri F, Noel LH, Stengel B, Even C, Kreis H, Mignon F, Grunfeld JP. Lithium-induced nephropathy: rate of progression and prognostic factors. Kidney Int 64: 585–592, 2003.[CrossRef][Web of Science][Medline]
  26. Rao R, Zhang MZ, Zhao M, Cai H, Harris RC, Breyer MD, Hao CM. Lithium treatment inhibits renal GSK-3 activity and promotes cyclooxygenase 2-dependent polyuriarats. Am J Physiol Renal Physiol 288: F642–F649, 2005.[Abstract/Free Full Text]
  27. Rindler MJ, Saier MH Jr. Evidence for Na+/H+ antiport in cultured dog kidney cells (MDCK). J Biol Chem 256: 10820–10825, 1981.[Abstract/Free Full Text]
  28. Rojek A, Nielsen J, Brooks HL, Gong H, Kim YH, Kwon TH, Frokær J, Nielsen S. Altered expression of selected genes in kidney of rats with lithium-induced NDI. Am J Physiol Renal Physiol 288: F1276–F1289, 2005.[Abstract/Free Full Text]
  29. Sands JM. Mammalian urea transporters. Annu Rev Physiol 65: 543–566, 2003.[CrossRef][Web of Science][Medline]
  30. Stefan E, Wiesner B, Baillie GS, Mollajew R, Henn V, Lorenz D, Furkert J, Santamaria K, Nedvetsky P, Hundsrucker C, Beyermann M, Krause E, Pohl P, Gall I, MacIntyre AN, Bachmann S, Houslay MD, Rosenthal W, Klussmann E. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol 18: 199–212, 2007.[Abstract/Free Full Text]
  31. Timmer RT, Sands JM. Lithium intoxication. J Am Soc Nephrol 10: 666–674, 1999.[Free Full Text]
  32. Walker RG. Lithium nephrotoxicity. Kidney Int Suppl 42: S93–S98, 1993.[Medline]
  33. Wall SM, Han JS, Chou CL, Knepper MA. Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD. Am J Physiol Renal Fluid Electrolyte Physiol 262: F989–F998, 1992.[Abstract/Free Full Text]
  34. Willoughby D, Wong W, Schaack J, Scott JD, Cooper DM. An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. EMBO J 25: 2051–2061, 2006.[CrossRef][Web of Science][Medline]
  35. Yamaki M, Kusano E, Tetsuka T, Takeda S, Homma S, Murayama N, Asano Y. Cellular mechanism of lithium-induced nephrogenic diabetes insipidus in rats. Am J Physiol Renal Fluid Electrolyte Physiol 261: F505–F511, 1991.[Abstract/Free Full Text]
  36. Yasui M, Zelenin SM, Celsi G, Aperia A. Adenylate cyclase-coupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements. Am J Physiol Renal Physiol 272: F443–F450, 1997.[Abstract/Free Full Text]
  37. Zhang C, Sands JM, Klein JD. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85–F90, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
A. C. Mistry, R. Mallick, J. D. Klein, T. Weimbs, J. M. Sands, and O. Frohlich
Syntaxin specificity of aquaporins in the inner medullary collecting duct
Am J Physiol Renal Physiol, August 1, 2009; 297(2): F292 - F300.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/3/F518    most recent
00349.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fröhlich, O.
Right arrow Articles by Sands, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fröhlich, O.
Right arrow Articles by Sands, J. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.