Posttranscriptional control of aquaporin-2 abundance by vasopressin in renal collecting duct principal cells
http://www.100md.com
《美国生理学杂志》
1Service of Nephrology, Fondation pour Recherches Medicales, Geneva, Switzerland
2The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark
ABSTRACT
Prevailing expression levels of aquaporin-2 (AQP2) mRNA play a major role in regulating AQP2 protein abundance. Here, we investigated whether AQP2 protein abundance is regulated at a posttranscriptional level as well. The expression levels of both AQP2 mRNA and protein increase in response to arginine vasopressin (AVP) in a concentration- and time-dependent manner in cultured immortalized mouse collecting duct principal cells (mpkCCDcl4 cells). AVP washout from the medium of AVP-pretreated cells revealed that AQP2 mRNA expression progressively decreased over time, whereas AQP2 protein abundance first increased immediately after AVP washout and then gradually decreased over time. Inversely, increasing AVP concentration led to a time-dependent increase of AQP2 mRNA, whereas AQP2 protein abundance first decreased immediately after AVP supplementation and then gradually increased over time. These transient effects arose from altered V2-receptor activity because they could be abolished by SR-121463B, a specific V2-receptor antagonist. Although cycloheximide administration had no effect on transient alterations of AQP2 protein content, these effects were attenuated by administration of chloroquine, a lysosomal inhibitor, or lactacystin, a proteasomal inhibitor. Short-term inhibition of PKA activity significantly increased AQP2 protein abundance and blunted the transient alterations of AQP2 protein content induced by AVP washout and supplementation. In addition, phosphorylated AQP2 abundance increased immediately after AVP supplementation. These results indicate that in response to AVP AQP2 protein abundance in collecting duct principal cells is principally influenced by AQP2 mRNA content but is additionally regulated by PKA-dependent negative feedback acting on AQP2 protein degradation.
arginine vasopressin; protein degradation
AQUAPORINS CONSTITUTE a family of proteins that facilitate water transport across cell membranes. In mammals, at least 12 members of the aquaporin (AQP) family have been identified so far, and at least 4 AQP isoforms (AQP1–AQP4) play a major role in renal water reabsorption (30). AQP1 is expressed in the proximal tubule and thin descending limb of Henle and mediates constitutive water reabsorption. AQP2–AQP4 are mainly expressed in the collecting duct (CD) where water enters the cell via apical AQP2 and exits the cell via basolateral AQP3 and AQP4. A feature that sets AQP2, and AQP3 to a lesser extent, apart from the other members of the AQP family is that its expression is largely dependent on the antidiuretic hormone [8-arginine]vasopressin (AVP), which plays a key role in regulated water excretion. Acute increases of circulating AVP concentration induce rapid apical cell surface expression of AQP2 that increases CD water permeability (28), whereas sustained increases of AVP concentration increase total AQP2 and AQP3 content, further enhancing CD water permeability (41). AVP controls AQP2 expression by binding to basolateral V2 receptors, an event that leads to Gs/adenylyl cyclase activation, increased cAMP concentration, and increased cAMP-dependent protein kinase activity (7). In addition to AVP, other factors also influence AQP2 protein content by regulating AQP2 mRNA expression. This has been shown in animals treated with V2-receptor antagonists (23, 26), which display increased AQP2 expression in response to water restriction. Reciprocally, an AVP-independent decrease of AQP2 expression has been shown as a consequence of water loading in AVP-supplemented animals (11, 26), lithium treatment (21, 24), and unilateral ureteral obstruction (12).
Numerous observations show coordinated expression levels occurring between AQP2 mRNA and protein, suggesting that AQP2 protein abundance is directly modulated via altered expression of AQP2 mRNA. For instance, long-term treatment with the V2-receptor agonist [1-deamino,8-D-arginine]vasopressin (dDAVP) results in increased expression of both AQP2 mRNA and protein in rats (11), whereas decreased levels of both AQP2 mRNA and protein were observed in rats treated with V2-receptor antagonists (18, 23, 35). Coordinated decreases or increases of AQP2 mRNA and protein expression were also observed in water-loaded and dehydrated rats, respectively (11, 18, 22, 23, 26). Of clinical relevance, parallel increased expression levels of AQP2 mRNA and protein reflecting increased AVP activity have been demonstrated in animal models of hepatic cirrhosis (3) and chronic heart failure (44) as well as in pregnant rats (33), whereas decreased expression levels were found in animals subjected to lithium treatment (21, 24), ureteral obstruction (12), or potassium deprivation (2, 25). However, uncoupled expression between AQP2 mRNA and protein has also been described under certain conditions. In hypercalcemic rats, polyuria was accompanied by decreased expression of AQP2 protein, but not mRNA, in the inner medullary collecting duct (IMCD) (38). Fasting was also shown to result in downregulated AQP2 protein expression in the cortex and outer medulla, although cortical AQP2 mRNA expression remained unaltered (1, 42). These observations suggest that, in addition to control exerted at the mRNA level, AQP2 protein expression may be adjusted at a posttranscriptional level as well.
We have previously shown that, when grown on permeable filters, mpkCCDcl4 cells, derived from microdissected cortical CDs of a SVPK/Tag transgenic mouse, express large quantities of AQP2 mRNA and protein in response to physiological concentrations of basolateral AVP (10–10 M) (5, 14–16). Interestingly, when AVP was washed from the cell medium, AQP2 protein expression transiently increased before AQP2 degradation (14). This observation suggests that in mpkCCDcl4 cells AVP controls AQP2 protein expression both at transcriptional and posttranscriptional levels. In the present study, we further investigated posttranscriptional AQP2 regulation and the mechanisms involved in this process.
MATERIALS AND METHODS
Cell culture. mpkCCDcl4 cells (passages 22–31) were grown in defined medium supplemented with 2% fetal calf serum (DM: DMEM-Ham’s F12 at 1:1 vol/vol, 60 nM sodium selenate, 5 μg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 20 mM D-glucose, and 20 mM HEPES, pH 7.4) (4) at 37°C in 5% CO2-95% air atmosphere. Experiments were performed on confluent cells seeded on permeable filters (Transwell, 0.4-μm pore size, 1-cm2 growth area; Corning Costar, Cambridge, MA). Cells were grown in DM until confluent and then in serum-free and hormone-deprived DM 24 h before experiments.
Western blot analysis. After incubation, mpkCCDcl4 cells were homogenized in 150 μl of ice-cold lysis buffer: 20 mM Tris·HCl, 2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mM Na4O7P2, 2 mM Na3VO4, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 10 μg/ml leupeptin, 4 μg/ml aprotinin, and 1% Triton X-100, pH 7.4. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). AQP2, pAQP2 (phosphorylated AQP2 at Ser-256), and Na-K-ATPase -subunit were detected by Western blotting using rabbit antibodies as described (6, 27, 44). The antigen-antibody complexes were detected by the SuperSignal substrate method (Pierce, Rockford, IL). Bands were quantified using a video densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Real-time PCR analysis. After incubation, total RNA from mpkCCDcl4 cells was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were measured by UV spectrophotometry. One microgram of RNA was used to synthesize cDNA using SuperScript II RNase H– reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR was performed on 10 μl of cDNA diluted 1:20 (vol/vol) using 3 ng of each primer and 12.5 μl of SYBR green Master Mix (Applied Biosystems) to obtain a final reaction volume of 25 μl. Triplicate amplification reactions were performed with an ABI Prism 7000 sequence detection system (Applied Biosystems). Primers used for detection of mouse acidic ribosomal phosphoprotein P0 were 5'-AATCTCCAGAGGCACCATTG-3' and 5'-GTTCAGCATGTTCAGCAGTG-3', those for AQP2 were 5'-CTTCCTTCGAGCTGCCTTC-3' and 5'-CATTGTTGTGGAGAGCATTGAC-3', and those for V2 receptor were 5'-CGTGGGATCCAGAAGCTCC-3' and 5'-GGCTAGCCAGCAGCATGA-3'. Data were analyzed using ABI Prism software (Applied Biosystems), and Po was used as an internal standard. Differences (fold) in cDNA abundance (F) were calculated using the formula F = 2(Ct1 – Ct2), where Ct1 and Ct2 are the number of cycles required to reach the threshold of amplicon abundance for experimental and control conditions, respectively.
Statistics. Results are given as means ± SE from n independent experiments. Each experiment was performed on mpkCCDcl4 cells from the same passage. Statistical differences were assessed using the Mann-Whitney U-test or the Kruskal-Wallis test for comparison of two groups or more, respectively. A P value <0.05 was considered significant.
RESULTS
AVP increases AQP2 mRNA and protein in a dose- and time-dependent manner in mpkCCDcl4 cells. We previously reported that the expression levels of endogenous AQP2 protein dramatically increase in mpkCCDcl4 cells grown on permeable filters in response to AVP added to the basal medium (14). In the present study, we compared the concentration dependence and the time course of the effects of AVP on AQP2 mRNA and protein expression in mpkCCDcl4 cells. AQP2 protein content was determined by Western blot analysis, where AQP2 protein was revealed by a narrow 29-kDa band and a more diffuse band of 45 kDa, representing the nonglycosylated and glycosylated forms of AQP2, respectively (33). AQP2 mRNA content was determined by real-time PCR analysis. Both AQP2 protein (Fig. 1, A and B) and mRNA (Fig. 1C) increased in a coordinated manner in response to 24 h of stimulation in the presence of increasing AVP concentrations with half-maximal and maximal expression occurring at 10–10 and 10–9 M AVP, respectively. Time course experiments revealed that the rise in expression levels of AQP2 protein (Fig. 1, D and E) was preceded by the rise of mRNA (Fig. 1F) in mpkCCDcl4 cells treated with 10–10 M AVP. Half-maximal AQP2 mRNA expression was observed after 3 h of stimulation, and maximal levels were obtained after 8 h, whereas AQP2 protein expression after 8 h of stimulation was only 20% of that obtained after 24 h, indicating that translation of AQP2 mRNA to protein requires several hours in AVP-stimulated mpkCCDcl4 cells.
AVP induces posttranscriptional processing of AQP2 protein in mpkCCDcl4 cells. We have previously shown that AQP2 protein expression increases in AVP-pretreated mpkCCDcl4 cells 1 h after AVP was washed from the medium (AVP chase), compared with AQP2 expression levels of cells that were continuously subjected to AVP (14). In the present study, we compared AQP2 mRNA and protein expression in mpkCCDcl4 cells that were first pretreated 24 h with AVP 10–10 M, after which time AVP was removed from the medium and incubated in the absence of AVP for variable periods of time. Compared with cells treated for 24 h with AVP, AQP2 protein expression significantly increased during the first 3 h of AVP chase and then decreased to reach near baseline levels after 24 h of AVP chase (Fig. 2, A and B). Contrary to AQP2 protein, AQP2 mRNA gradually decreased over the entire period of AVP chase (Fig. 2C). Because AVP chase led to increased levels of AQP2 protein expression shortly after AVP washout, we wondered whether these results could be mirrored by abruptly increasing the AVP concentration in AVP-pretreated cells. This was investigated by administrating 10–9 M AVP to cells pretreated 24 h with 10–10 M AVP and by following AQP2 mRNA and protein expression over time. AQP2 protein expression was found to decrease during the first hour after addition of 10–9 M AVP to the cell medium, after which time it gradually increased (Fig. 2, D and E). Contrary to AQP2 protein, AQP2 mRNA expression gradually increased over the entire period after addition of 10–9 M AVP to the cell medium (Fig. 2F).
The specificity of AVP washout acting on AQP2 expression and the role played by the V2 receptor in this process were investigated. Real-time PCR analysis revealed high V2-receptor mRNA content (cycle threshold for V2 receptor was <26 and that for Po was <18; these values were not significantly altered by 24 h of 10–10 M AVP stimulation or by either 30 min of AVP chase or 30 min of 10–9 M AVP stimulation), indicating stable expression of this receptor mRNA in mpkCCDcl4 cells. Expression levels of AQP2 protein were similarly increased in cells subjected 30 min to AVP chase and cells subjected to 10–8 M SR-121463B, a nonpeptidic, competitive, and specific V2-receptor antagonist (39), whereas addition of 10–10 M AVP immediately after AVP washout blunted the increase of AQP2 protein expression compared with cells subjected for 24 h to 10–10 M AVP (Fig. 3, A and B). These results indicate that upregulated AQP2 protein expression shortly after AVP washout results from blunted V2-receptor activity and not from replacement of the incubation medium. To examine the role played by the V2 receptor in downregulated AQP2 protein expression that occurs shortly after 10–9 M AVP administration, 10–10 M AVP-pretreated cells were treated with 10–9 M dDAVP for 30 min. Both AVP and dDAVP decreased AQP2 protein expression to similar extents (Fig. 3, C and D, lanes 1–3), indicating that the V2 receptor plays a key role in this process. This was further confirmed by treating AVP-pretreated cells with 10–8 M SR-121463B for 30 min followed by an additional 30 min of incubation in the presence of 10–9 M AVP. Although 10–9 M AVP decreased AQP2 protein expression in the absence of SR-121463B, the increased expression levels of AQP2 protein induced by SR-121463B were not altered by 10–9 M AVP (Fig. 3, C and D, lanes 4–7).
Together, the time course experiments performed in 10–10 M AVP-pretreated cells subjected to AVP chase or to 10–9 M AVP suggest that V2-receptor activity mediates posttranscriptional inhibition of AQP2 protein expression in mpkCCDcl4 cells. Such a negative feedback mechanism induced by AVP would attenuate expression of AQP2 protein.
AVP enhances AQP2 protein degradation in mpkCCDcl4 cells. An AVP-induced mobilization of sarcoplasmic/endoplasmic reticulum Ca2+ stores that leads to attenuated protein synthesis during the first 30 min after AVP stimulation via the V1 receptor has been demonstrated in H9c2 ventricular myocytes (37). AVP-induced intracellular Ca2+ mobilization via stimulation of V2 receptors has also been demonstrated in perfused rat renal CD (9, 45), an event that participates in regulated AQP2 trafficking (9). Because BAPTA has been shown to block both the AVP-induced increase in intracellular free Ca2+ and the ensuing increase of osmotic water permeability (9, 45), we tested whether the decreased levels of AQP2 content shortly after 10–9 M AVP administration may be due, at least in part, to intracellular Ca2+ mobilization. For this purpose, 10–10 M AVP-pretreated mpkCCDcl4 cells were treated with 10–5 M BAPTA-AM for 1 h prior to 30 min of 10–9 M AVP stimulation. The extent of the decrease of AQP2 protein content was similar between cells treated or not with BAPTA-AM (Fig. 4, A and B, compare decreased expression between lanes 1 and 2 and between lanes 3 and 4), indicating that decreased AQP2 protein expression following enhanced AVP stimulation is not due to intracellular Ca2+ mobilization.
We further examined the role that altered translational activity may play in the increased or decreased expression levels of AQP2 protein that occur shortly after AVP chase and 10–9 AVP administration, respectively. We investigated altered AQP2 mRNA translation by treating 10–10 M AVP-pretreated mpkCCDcl4 cells for 24 h with 2 x 10–5 M of the translational inhibitor cycloheximide for 30 min before 30 min of AVP chase or 10–9 M AVP stimulation. Cells treated with cycloheximide expressed significantly less AQP2 protein than cells treated with 10–10 M AVP alone (Fig. 4, C and D, compare lanes 4 and 1), reflecting reduced synthesis of AQP2 protein. Importantly, increased AQP2 protein content induced by 30 min of AVP chase was not different between cells treated or not with cycloheximide (Fig. 4, C and D, compare lanes 2 and 5). Moreover, the extent of decreased AQP2 protein content induced by 30 min of 10–9 M AVP stimulation was similar between cells treated or not with cycloheximide (Fig. 4, C and D, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). These results strongly suggest that AQP2 mRNA translation in mpkCCDcl4 cells is not altered immediately after altered V2-receptor activity.
We have previously shown that both lysosomal and proteasomal pathways participate in AQP2 protein degradation in mpkCCDcl4 cells (14). We examined AQP2 protein degradation shortly following AVP removal or AVP supplementation by treating 10–10 M AVP-pretreated cells with a lysomal or proteasomal inhibitor for 8 h, after which period AVP was washed from the medium or readministered at a concentration of 10–9 M for an additional 30 min. Chloroquine (10–4 M), a weak base that increases lysosomal pH thereby inhibiting the proteolytic activity of lysosomal enzymes, increased AQP2 protein content compared with untreated cells (Fig. 5, A and B, compare lanes 4 and 1). Results show that the increase of AQP2 protein content induced after 30 min of AVP chase was similar between cells treated or not with chloroquine (Fig. 5, A and B, compare lanes 2 and 5). In addition, the decrease in AQP2 protein content induced after 30 min by 10–9 M AVP was attenuated in cells treated with chloroquine (Fig. 5, A and B, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). Administration of 5 x 10–6 M lactacystin, a potent and specific inhibitor of the proteasomal pathway, produced results similar to chloroquine. Lactacystin increased AQP2 protein content compared with untreated cells (Fig. 5, C and D, compare lanes 4 and 1). Results show that the extent of increased AQP2 protein expression induced after 30 min of AVP chase was similar between cells treated or not with lactacystin (Fig. 5, C and D, compare lanes 2 and 5). In addition, lactacytin attenuated decreased AQP2 protein expression induced after 30 min by 10–9 M AVP (Fig. 5, C and D, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). Together, our results reveal a transient decrease of AQP2 protein degradation shortly following abrogated V2-receptor activity and increased AQP2 degradation shortly following enhanced V2-receptor stimulation. In addition, our results suggest that both lysosomal and proteasomal pathways participate in this process.
The intracellular mechanisms that participate in the transient alterations of AQP2 protein content following altered V2-receptor activity was further investigated in mpkCCDcl4 cells. Modulation of renal water transport by PGE2 is well established (13) and at least partly results from AVP antagonism. PGE2 was additionally shown to reverse AVP-induced AQP2 translocation to the plasma membrane (46) and inhibition of cyclooxygenase-2 (COX-2), a key player in PGE2 synthesis, prevented downregulated AQP2 expression induced by bilateral urelateral obstruction (8, 31). Because COX-2 expression is induced by AVP in the inner medulla (47), we investigated whether the transient alterations of AQP2 protein expression after AVP removal or supplementation are linked to reduced or enhanced PGE2 synthesis. This was performed by comparing altered AQP2 protein expression between cells treated or not with 10–5 M indomethacin, a classical COX inhibitor. As shown in Fig. 6, indomethacin did not significantly alter the transient increase or decrease of AQP2 protein content, indicating that altered PGE2 synthesis is not responsible for this event.
We next investigated the role of PKA activity in AVP-inducible AQP2 posttranscriptional processing by comparing altered AQP2 protein expression between cells treated or not with 5 x 10–6 M myristoylated PKA inhibitor (PKI; Calbiochem). The efficacy of PKI in mpkCCDcl4 cells was previously confirmed (16). One hour of PKI treatment significantly increased AQP2 protein abundance (Fig. 7, A and B, compare lanes 4 and 1), blunted AQP2 protein upregulation induced by AVP washout (Fig. 7, A and B, compare increased expression between lanes 1 and 2 and between lanes 4 and 5), and attenuated AQP2 protein downregulation induced by AVP supplementation (Fig. 7, A and B, compare reduced expression between lanes 1 and 3 and between lanes 4 and 6). These results strongly suggest that the transient changes of AQP2 protein abundance, and therefore degradation, resulting from altered V2-receptor activity in mpkCCDcl4 cells are largely mediated by changes in PKA activity. This incited us to investigate whether altered V2-receptor activity influences phosphorylation of AQP2 itself since AQP2 phosphorylation, at Ser-256, is mediated by PKA (20). For this purpose, we performed Western blot analysis using antiserum against AQP2 amino acids 253–262 containing phosphorylated Ser-256 to measure pAQP2 levels after 30 min of AVP chase or AVP supplementation in AVP-pretreated cells. Similar to total AQP2, i.e., the AQP2 signal obtained by the polyclonal anti-AQP2 antibody (44) (Fig. 7, C and D), the intensity of the signal corresponding to pAQP2, revealed by several distinct bands 29 kDa in size and a more diffuse band 45 kDa in size, increased in an AVP dose- and time-dependent manner (Fig. 7, E and F, compare lanes 6, 3, and 1 and compare lanes 1 and 2). Interestingly, although 30 min of AVP chase increased total AQP2 and pAQP2 abundance proportionally (Fig. 7, E and F, compare lanes 3 and 4), 30 min of AVP supplementation increased pAQP2 content (Fig. 7, E and F, compare lanes 3 and 5) despite decreased abundance of total AQP2 (Fig. 7, C and D, compare lanes 4 and 6). This observation indicates that AQP2 phosphorylation at Ser-256 may precede AQP2 degradation induced by AVP. Close inspection of pAQP2 immunoblots revealed two bands corresponding to 28 and 29 kDa, discernable in the presence of AVP. These two bands were replaced by a single band of 28 kDa in response to 30 min of AVP chase. On the other hand, 30 min of 10–9 M AVP supplementation resulted in a shift toward the 29-kDa band. We hypothesize that the 29-kDa band may result from posttranslational processing and may represent an AVP-induced AQP2 predegradation product.
DISCUSSION
We have previously shown that stimulation of mpkCCDcl4 cells with AVP induces upregulated expression of both AQP2 mRNA and protein, whereas removal of AVP from the cell medium ultimately leads to their degradation (14). In the present study, AVP washout was found to transiently increase AQP2 protein expression, whereas enhanced V2-receptor activity mirrored this effect by transiently decreasing AQP2 protein expression. The altered expression levels of AQP2 protein were not associated with altered expression of AQP2 mRNA, indicating that in addition to transcriptional control AQP2 protein expression is also regulated at a posttranscriptional level in mpkCCDcl4 cells. Posttranscriptional processing of AQP2 protein was found to depend on AQP2 protein degradation rather than altered protein synthesis, a process involving both lysosomal and proteasomal degradation pathways. Together, our results suggest that in cultured mpkCCDcl4 cells AVP induces both AQP2 gene transcription and enhances AQP2 protein degradation. The physiological context of AVP-mediated increased degradation of AQP2 protein shown in the present study may be comparable to that of investigations that demonstrate conditional protein degradation of specific targets induced by specific stimuli. For instance, lysosomal degradation of E-cadherin induced by v-Src was found to participate in cell-to-cell dissociation during epithelial to mesenchymal transition (34), and proteasome-dependent degradation of the transcription factor AP-2 by TNF- was found to play a critical role in TNF--induced apoptosis (32).
Most reports from literature indicate coordinated expression levels between AQP2 mRNA and protein. For instance, the expression levels of both AQP2 mRNA and protein increased in rats treated with AVP (17, 29) and decreased in rats treated with V2-receptor antagonists (10, 23, 26). These observations suggest that AQP2 gene transcription, and possibly selective degradation of AQP2 mRNA, plays a capital role in controlled AQP2 protein abundance. However, under certain conditions, uncoupled expression between AQP2 protein and mRNA has been described. The enhanced water permeability of isolated IMCD from rats made chronically hypercalcemic by dihydrotachysterol was accompanied by significantly reduced expression levels of AQP2 protein, but not by mRNA (38). Polyuria exhibited in rats deprived of food for 24 h was also shown to be accompanied by decreased AQP2 protein, but not mRNA, content in the cortex, whereas the outer medulla displayed decreased expression of both AQP2 protein and mRNA (1, 42). These observations are indicative of AQP2 posttranscriptional processing occurring in vivo.
Further indications of in vivo regulation of AQP2 protein content occurring at a posttranscriptional level has been provided by experiments performed with V2-receptor antagonists. Administration of OPC-31260 in rats revealed decreased AQP2 mRNA in inner medulla expression occurring within 30 min of treatment, which persisted 24 h later (10). AQP2 protein content, however, was found to be similar between OPC-31260- and sham-operated animals after 1 and 24 h of treatment and only decreased in animals administered with OPC-31260 for 60 h (10, 23). The lag of AQP2 protein degradation compared with that of AQP2 mRNA may possibly be due to slow AQP2 protein degradation occurring in these animals, as opposed to rapid degradation of AQP2 mRNA. However, rapidly downregulated expression of AQP2 protein has been described under certain conditions. For instance, rats fed a K+-free diet displayed decreased cortical AQP2 mRNA and protein abundance after 12 h of K+ deprivation (2). Downregulated expression of both AQP2 mRNA and protein was also observed after 24 h in inner medulla of obstructed kidneys (12). Because both of these conditions do not arise from decreased plasma AVP concentration (12, 25, 43), it is possible that reduced V2-receptor activity in OPC-31260-treated animals temporally reduces AQP2 protein degradation, an event that occurs concurrently with decreased AQP2 mRNA expression. This effect would be similar to that found in the present study produced in cultured mpkCCDcl4 cells in which AVP chase or SR-121463B administration transiently increased AQP2 protein, but not mRNA, abundance via decreased AQP2 protein degradation.
Interestingly, the decreased abundance of AQP2 protein, but not mRNA, in both dihydrotachysterol-treated and fasted animals occurred independently of changes in AVP plasma concentration (36, 42). Similar changes in AQP2 expression resulting from fasting were also observed in AVP-deficient Brattleboro rats (42). Together with the results of the present study, these observations indicate that posttranscriptional changes in AQP2 protein content may be induced by AVP and/or by factors acting independently of AVP, possibly by acting via pathways normally stimulated by AVP and that influence protein degradation. Although recent studies have demonstrated decreased AQP2 abundance resulting from increased COX activity (8, 31), our results indicate that, in mpkCCDcl4 cells, this pathway is not responsible for altered AQP2 protein abundance occurring in response to acute variations of V2-receptor activity.
The results of the present study suggest that PKA controls AQP2 abundance at two distinct levels: it promotes AQP2 mRNA expression on the one hand and retroactively constrains AQP2 protein abundance on the other hand, most likely by promoting AQP2 degradation. The rapid (30 min) effects produced by AVP washout and AVP supplementation on AQP2 protein abundance additionally suggest that the mechanism responsible for these effects consists of posttranscriptional processing, i.e., phosphorylation, as opposed to transcriptional-mediated processing. This is further supported by the observation that both effects occur regardless of the presence or absence of the translational inhibitor cycloheximide. The observation that pAQP2 abundance is increased after 30 min of AVP supplementation indicates that AQP2 degradation is preceded by AQP2 phosphorylation at Ser-256. AQP2 phosphorylation is necessary for its cell surface expression (19). The observation made by the present study that links PKA activity to AVP-induced AQP2 protein degradation, possibly via AQP2 phosphorylation, in mpkCCDcl4 cells may be tentatively compared with observations made in renal CD8 cells and IMCD cells, establishing the necessity of active PKA for AQP2 cell surface expression, a process that involves phosphorylation of AQP2 itself and of other proteins that mediate AQP2 exocytosis, such as RhoA (27, 40). Although further investigation is needed, these observations indicate that AQP2 cell surface expression may be tightly regulated by PKA via dual stimulation of AQP2 exocytosis and degradation.
In conclusion, our results indicate that AQP2 protein abundance is regulated at a posttranscriptional level via increased AQP2 protein degradation induced by enhanced V2-receptor activity. This process is dependent on PKA activity and involves phosphorylation of AQP2 itself. PKA may therefore play a dual role in controlling AQP2 abundance: it increases AQP2 gene transcription following V2-receptor stimulation and retroactively controls AQP2 protein content by promoting its degradation.
GRANTS
This work was supported by the Swiss National Foundation for Science Grant 3100-067878.02, by a grant from the Carlos et Elsie De Reuter Foundation to E. Feraille, and by a grant from the Novartis Foundation to U. Hasler.
FOOTNOTES
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.
E. Feraille and P.-Y. Martin contributed equally to this work.
REFERENCES
Amlal H, Chen Q, Habo K, Wang Z, and Soleimani M. Fasting downregulates renal water channel AQP2 and causes polyuria. Am J Physiol Renal Physiol 280: F513–F523, 2001.
Amlal H, Krane CM, Chen Q, and Soleimani M. Early polyuria and urinary concentrating defect in potassium deprivation. Am J Physiol Renal Physiol 279: F655–F663, 2000.
Asahina Y, Izumi N, Enomoto N, Sasaki S, Fushimi K, Marumo F, and Sato C. Increased gene expression of water channel in cirrhotic rat kidneys. Hepatology 21: 169–173, 1995.
Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, and Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol 10: 923–934, 1999.
Bustamante M, Hasler U, Kotova O, Chibalin AV, Mordasini D, Rousselot M, Vandewalle A, Martin PY, and Feraille E. Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells. Am J Physiol Renal Physiol 288: F334–F344, 2005.
Carranza ML, Feraille E, and Favre H. Protein kinase C-dependent phosphorylation of Na+-K+-ATPase -subunit in rat kidney cortical tubules. Am J Physiol Cell Physiol 271: C136–C143, 1996.
Chabardes D, Firsov D, Aarab L, Clabecq A, Bellanger AC, Siaume-Perez S, and Elalouf JM. Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content. J Biol Chem 271: 19264–19271, 1996.
Cheng X, Zhang H, Lee HL, and Park JM. Cyclooxygenase-2 inhibitor preserves medullary aquaporin-2 expression and prevents polyuria after ureteral obstruction. J Urol 172: 2387–2390, 2004.
Chou CL, Yip KP, Michea L, Kador K, Ferraris JD, Wade JB, and Knepper MA. Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin. J Biol Chem 275: 36839–36846, 2000.
Christensen BM, Marples D, Jensen UB, Frokiaer J, Sheikh-Hamad D, Knepper M, and Nielsen S. Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution. Am J Physiol Renal Physiol 275: F285–F297, 1998.
Ecelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, and Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 1852–1863, 1997.
Frokiaer J, Christensen BM, Marples D, Djurhuus JC, Jensen UB, Knepper MA, and Nielsen S. Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction. Am J Physiol Renal Physiol 273: F213–F223, 1997.
Harris RC and Breyer MD. Physiological regulation of cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol 281: F1–F11, 2001.
Hasler U, Mordasini D, Bens M, Bianchi M, Cluzeaud F, Rousselot M, Vandewalle A, Feraille E, and Martin PY. Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells. J Biol Chem 277: 10379–10386, 2002.
Hasler U, Mordasini D, Bianchi M, Vandewalle A, Feraille E, and Martin PY. Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells. J Biol Chem 278: 21639–21648, 2003.
Hasler U, Vinciguerra M, Vandewalle A, Martin PY, and Feraille E. Dual effects of hypertonicity on aquaporin-2 expression in cultured renal collecting duct principal cells. J Am Soc Nephrol 16: 1571–1582, 2005.
Hayashi M, Sasaki S, Tsuganezawa H, Monkawa T, Kitajima W, Konishi K, Fushimi K, Marumo F, and Saruta T. Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney. J Clin Invest 94: 1778–1783, 1994.
Hayashi M, Sasaki S, Tsuganezawa H, Monkawa T, Kitajima W, Konishi K, Fushimi K, Marumo F, and Saruta T. Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney. J Clin Invest 94: 1778–1783, 1994.
Kamsteeg EJ, Heijnen I, van Os CH, and Deen PM. The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers. J Cell Biol 151: 919–930, 2000.
Kuwahara M, Fushimi K, Terada Y, Bai L, Marumo F, and Sasaki S. cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J Biol Chem 270: 10384–10387, 1995.
Laursen UH, Pihakaski-Maunsbach K, Kwon TH, Ostergaard Jensen E, Nielsen S, and Maunsbach AB. Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus. Nephron Exp Nephrol 97: 1–16, 2004.
Ma T, Hasegawa H, Skach WR, Frigeri A, and Verkman AS. Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. Am J Physiol Cell Physiol 266: C189–C197, 1994.
Marples D, Christensen BM, Frokiaer J, Knepper MA, and Nielsen S. Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats. Am J Physiol Renal Physiol 275: F400–F409, 1998.
Marples D, Christensen S, Christensen EI, Ottosen PD, and Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 95: 1838–1845, 1995.
Marples D, Frokiaer J, Dorup J, Knepper MA, and Nielsen S. Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. J Clin Invest 97: 1960–1968, 1996.
Murillo-Carretero MI, Ilundain AA, and Echevarria M. Regulation of aquaporin mRNA expression in rat kidney by water intake. J Am Soc Nephrol 10: 696–703, 1999.
Nejsum LN, Zelenina M, Aperia A, Frokiaer J, and Nielsen S. Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation. Am J Physiol Renal Physiol 288: F930–F938, 2005.
Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, and 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.
Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663–11667, 1993.
Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, and Knepper MA. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205–244, 2002.
Norregaard R, Jensen BL, Li C, Wang W, Knepper MA, Nielsen S, and Frokiaer J. COX-2 inhibition prevents downregulation of key renal water and sodium transport proteins in response to bilateral ureteral obstruction. Am J Physiol Renal Physiol 289: F322–F333, 2005.
Nyormoi O, Wang Z, Doan D, Ruiz M, McConkey D, and Bar-Eli M. Transcription factor AP-2 is preferentially cleaved by caspase 6 and degraded by proteasome during tumor necrosis factor -induced apoptosis in breast cancer cells. Mol Cell Biol 21: 4856–4867, 2001.
Ohara M, Martin PY, Xu DL, St. John J, Pattison TA, Kim JK, and Schrier RW. Upregulation of aquaporin 2 water channel expression in pregnant rats. J Clin Invest 101: 1076–1083, 1998.
Palacios F, Tushir JS, Fujita Y, and D’Souza-Schorey C. Lysosomal targeting of E-cadherin: a unique mechanism for the down-regulation of cell-cell adhesion during epithelial to mesenchymal transitions. Mol Cell Biol 25: 389–402, 2005.
Promeneur D, Kwon TH, Frokiaer J, Knepper MA, and Nielsen S. Vasopressin V2-receptor-dependent regulation of AQP2 expression in Brattleboro rats. Am J Physiol Renal Physiol 279: F370–F382, 2000.
Puliyanda DP, Ward DT, Baum MA, Hammond TG, and Harris HW Jr. Calpain-mediated AQP2 proteolysis in inner medullary collecting duct. Biochem Biophys Res Commun 303: 52–58, 2003.
Reilly BA, Brostrom MA, and Brostrom CO. Regulation of protein synthesis in ventricular myocytes by vasopressin. The role of sarcoplasmic/endoplasmic reticulum Ca2+ stores. J Biol Chem 273: 3747–3755, 1998.
Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, and Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978–F985, 1998.
Serradeil-Le Gal C, Lacour C, Valette G, Garcia G, Foulon L, Galindo G, Bankir L, Pouzet B, Guillon G, Barberis C, Chicot D, Jard S, Vilain P, Garcia C, Marty E, Raufaste D, Brossard G, Nisato D, Maffrand JP, and Le Fur G. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729–2738, 1996.
Tamma G, Klussmann E, Maric K, Aktories K, Svelto M, Rosenthal W, and Valenti G. Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells. Am J Physiol Renal Physiol 281: F1092–F1101, 2001.
Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414–F422, 1996.
Wilke C, Sheriff S, Soleimani M, and Amlal H. Vasopressin-independent regulation of collecting duct aquaporin-2 in food deprivation. Kidney Int 67: 201–216, 2005.
Wilson DR. Pathophysiology of obstructive nephropathy. Kidney Int 18: 281–292, 1980.
Xu DL, Martin PY, Ohara M, St John J, Pattison T, Meng X, Morris K, Kim JK, and Schrier RW. Upregulation of aquaporin-2 water channel expression in chronic heart failure rat. J Clin Invest 99: 1500–1505, 1997.
Yip KP. Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct. J Physiol 538: 891–899, 2002.
Zelenina M, Christensen BM, Palmer J, Nairn AC, Nielsen S, and Aperia A. Prostaglandin E2 interaction with AVP: effects on AQP2 phosphorylation and distribution. Am J Physiol Renal Physiol 278: F388–F394, 2000.
Zhang MZ, Sanchez Lopez P, McKanna JA, and Harris RC. Regulation of cyclooxygenase expression by vasopressin in rat renal medulla. Endocrinology 145: 1402–1409, 2004.(Udo Hasler, Sren Nielsen, Eric Feraille,)
2The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark
ABSTRACT
Prevailing expression levels of aquaporin-2 (AQP2) mRNA play a major role in regulating AQP2 protein abundance. Here, we investigated whether AQP2 protein abundance is regulated at a posttranscriptional level as well. The expression levels of both AQP2 mRNA and protein increase in response to arginine vasopressin (AVP) in a concentration- and time-dependent manner in cultured immortalized mouse collecting duct principal cells (mpkCCDcl4 cells). AVP washout from the medium of AVP-pretreated cells revealed that AQP2 mRNA expression progressively decreased over time, whereas AQP2 protein abundance first increased immediately after AVP washout and then gradually decreased over time. Inversely, increasing AVP concentration led to a time-dependent increase of AQP2 mRNA, whereas AQP2 protein abundance first decreased immediately after AVP supplementation and then gradually increased over time. These transient effects arose from altered V2-receptor activity because they could be abolished by SR-121463B, a specific V2-receptor antagonist. Although cycloheximide administration had no effect on transient alterations of AQP2 protein content, these effects were attenuated by administration of chloroquine, a lysosomal inhibitor, or lactacystin, a proteasomal inhibitor. Short-term inhibition of PKA activity significantly increased AQP2 protein abundance and blunted the transient alterations of AQP2 protein content induced by AVP washout and supplementation. In addition, phosphorylated AQP2 abundance increased immediately after AVP supplementation. These results indicate that in response to AVP AQP2 protein abundance in collecting duct principal cells is principally influenced by AQP2 mRNA content but is additionally regulated by PKA-dependent negative feedback acting on AQP2 protein degradation.
arginine vasopressin; protein degradation
AQUAPORINS CONSTITUTE a family of proteins that facilitate water transport across cell membranes. In mammals, at least 12 members of the aquaporin (AQP) family have been identified so far, and at least 4 AQP isoforms (AQP1–AQP4) play a major role in renal water reabsorption (30). AQP1 is expressed in the proximal tubule and thin descending limb of Henle and mediates constitutive water reabsorption. AQP2–AQP4 are mainly expressed in the collecting duct (CD) where water enters the cell via apical AQP2 and exits the cell via basolateral AQP3 and AQP4. A feature that sets AQP2, and AQP3 to a lesser extent, apart from the other members of the AQP family is that its expression is largely dependent on the antidiuretic hormone [8-arginine]vasopressin (AVP), which plays a key role in regulated water excretion. Acute increases of circulating AVP concentration induce rapid apical cell surface expression of AQP2 that increases CD water permeability (28), whereas sustained increases of AVP concentration increase total AQP2 and AQP3 content, further enhancing CD water permeability (41). AVP controls AQP2 expression by binding to basolateral V2 receptors, an event that leads to Gs/adenylyl cyclase activation, increased cAMP concentration, and increased cAMP-dependent protein kinase activity (7). In addition to AVP, other factors also influence AQP2 protein content by regulating AQP2 mRNA expression. This has been shown in animals treated with V2-receptor antagonists (23, 26), which display increased AQP2 expression in response to water restriction. Reciprocally, an AVP-independent decrease of AQP2 expression has been shown as a consequence of water loading in AVP-supplemented animals (11, 26), lithium treatment (21, 24), and unilateral ureteral obstruction (12).
Numerous observations show coordinated expression levels occurring between AQP2 mRNA and protein, suggesting that AQP2 protein abundance is directly modulated via altered expression of AQP2 mRNA. For instance, long-term treatment with the V2-receptor agonist [1-deamino,8-D-arginine]vasopressin (dDAVP) results in increased expression of both AQP2 mRNA and protein in rats (11), whereas decreased levels of both AQP2 mRNA and protein were observed in rats treated with V2-receptor antagonists (18, 23, 35). Coordinated decreases or increases of AQP2 mRNA and protein expression were also observed in water-loaded and dehydrated rats, respectively (11, 18, 22, 23, 26). Of clinical relevance, parallel increased expression levels of AQP2 mRNA and protein reflecting increased AVP activity have been demonstrated in animal models of hepatic cirrhosis (3) and chronic heart failure (44) as well as in pregnant rats (33), whereas decreased expression levels were found in animals subjected to lithium treatment (21, 24), ureteral obstruction (12), or potassium deprivation (2, 25). However, uncoupled expression between AQP2 mRNA and protein has also been described under certain conditions. In hypercalcemic rats, polyuria was accompanied by decreased expression of AQP2 protein, but not mRNA, in the inner medullary collecting duct (IMCD) (38). Fasting was also shown to result in downregulated AQP2 protein expression in the cortex and outer medulla, although cortical AQP2 mRNA expression remained unaltered (1, 42). These observations suggest that, in addition to control exerted at the mRNA level, AQP2 protein expression may be adjusted at a posttranscriptional level as well.
We have previously shown that, when grown on permeable filters, mpkCCDcl4 cells, derived from microdissected cortical CDs of a SVPK/Tag transgenic mouse, express large quantities of AQP2 mRNA and protein in response to physiological concentrations of basolateral AVP (10–10 M) (5, 14–16). Interestingly, when AVP was washed from the cell medium, AQP2 protein expression transiently increased before AQP2 degradation (14). This observation suggests that in mpkCCDcl4 cells AVP controls AQP2 protein expression both at transcriptional and posttranscriptional levels. In the present study, we further investigated posttranscriptional AQP2 regulation and the mechanisms involved in this process.
MATERIALS AND METHODS
Cell culture. mpkCCDcl4 cells (passages 22–31) were grown in defined medium supplemented with 2% fetal calf serum (DM: DMEM-Ham’s F12 at 1:1 vol/vol, 60 nM sodium selenate, 5 μg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 20 mM D-glucose, and 20 mM HEPES, pH 7.4) (4) at 37°C in 5% CO2-95% air atmosphere. Experiments were performed on confluent cells seeded on permeable filters (Transwell, 0.4-μm pore size, 1-cm2 growth area; Corning Costar, Cambridge, MA). Cells were grown in DM until confluent and then in serum-free and hormone-deprived DM 24 h before experiments.
Western blot analysis. After incubation, mpkCCDcl4 cells were homogenized in 150 μl of ice-cold lysis buffer: 20 mM Tris·HCl, 2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mM Na4O7P2, 2 mM Na3VO4, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 10 μg/ml leupeptin, 4 μg/ml aprotinin, and 1% Triton X-100, pH 7.4. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). AQP2, pAQP2 (phosphorylated AQP2 at Ser-256), and Na-K-ATPase -subunit were detected by Western blotting using rabbit antibodies as described (6, 27, 44). The antigen-antibody complexes were detected by the SuperSignal substrate method (Pierce, Rockford, IL). Bands were quantified using a video densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Real-time PCR analysis. After incubation, total RNA from mpkCCDcl4 cells was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were measured by UV spectrophotometry. One microgram of RNA was used to synthesize cDNA using SuperScript II RNase H– reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR was performed on 10 μl of cDNA diluted 1:20 (vol/vol) using 3 ng of each primer and 12.5 μl of SYBR green Master Mix (Applied Biosystems) to obtain a final reaction volume of 25 μl. Triplicate amplification reactions were performed with an ABI Prism 7000 sequence detection system (Applied Biosystems). Primers used for detection of mouse acidic ribosomal phosphoprotein P0 were 5'-AATCTCCAGAGGCACCATTG-3' and 5'-GTTCAGCATGTTCAGCAGTG-3', those for AQP2 were 5'-CTTCCTTCGAGCTGCCTTC-3' and 5'-CATTGTTGTGGAGAGCATTGAC-3', and those for V2 receptor were 5'-CGTGGGATCCAGAAGCTCC-3' and 5'-GGCTAGCCAGCAGCATGA-3'. Data were analyzed using ABI Prism software (Applied Biosystems), and Po was used as an internal standard. Differences (fold) in cDNA abundance (F) were calculated using the formula F = 2(Ct1 – Ct2), where Ct1 and Ct2 are the number of cycles required to reach the threshold of amplicon abundance for experimental and control conditions, respectively.
Statistics. Results are given as means ± SE from n independent experiments. Each experiment was performed on mpkCCDcl4 cells from the same passage. Statistical differences were assessed using the Mann-Whitney U-test or the Kruskal-Wallis test for comparison of two groups or more, respectively. A P value <0.05 was considered significant.
RESULTS
AVP increases AQP2 mRNA and protein in a dose- and time-dependent manner in mpkCCDcl4 cells. We previously reported that the expression levels of endogenous AQP2 protein dramatically increase in mpkCCDcl4 cells grown on permeable filters in response to AVP added to the basal medium (14). In the present study, we compared the concentration dependence and the time course of the effects of AVP on AQP2 mRNA and protein expression in mpkCCDcl4 cells. AQP2 protein content was determined by Western blot analysis, where AQP2 protein was revealed by a narrow 29-kDa band and a more diffuse band of 45 kDa, representing the nonglycosylated and glycosylated forms of AQP2, respectively (33). AQP2 mRNA content was determined by real-time PCR analysis. Both AQP2 protein (Fig. 1, A and B) and mRNA (Fig. 1C) increased in a coordinated manner in response to 24 h of stimulation in the presence of increasing AVP concentrations with half-maximal and maximal expression occurring at 10–10 and 10–9 M AVP, respectively. Time course experiments revealed that the rise in expression levels of AQP2 protein (Fig. 1, D and E) was preceded by the rise of mRNA (Fig. 1F) in mpkCCDcl4 cells treated with 10–10 M AVP. Half-maximal AQP2 mRNA expression was observed after 3 h of stimulation, and maximal levels were obtained after 8 h, whereas AQP2 protein expression after 8 h of stimulation was only 20% of that obtained after 24 h, indicating that translation of AQP2 mRNA to protein requires several hours in AVP-stimulated mpkCCDcl4 cells.
AVP induces posttranscriptional processing of AQP2 protein in mpkCCDcl4 cells. We have previously shown that AQP2 protein expression increases in AVP-pretreated mpkCCDcl4 cells 1 h after AVP was washed from the medium (AVP chase), compared with AQP2 expression levels of cells that were continuously subjected to AVP (14). In the present study, we compared AQP2 mRNA and protein expression in mpkCCDcl4 cells that were first pretreated 24 h with AVP 10–10 M, after which time AVP was removed from the medium and incubated in the absence of AVP for variable periods of time. Compared with cells treated for 24 h with AVP, AQP2 protein expression significantly increased during the first 3 h of AVP chase and then decreased to reach near baseline levels after 24 h of AVP chase (Fig. 2, A and B). Contrary to AQP2 protein, AQP2 mRNA gradually decreased over the entire period of AVP chase (Fig. 2C). Because AVP chase led to increased levels of AQP2 protein expression shortly after AVP washout, we wondered whether these results could be mirrored by abruptly increasing the AVP concentration in AVP-pretreated cells. This was investigated by administrating 10–9 M AVP to cells pretreated 24 h with 10–10 M AVP and by following AQP2 mRNA and protein expression over time. AQP2 protein expression was found to decrease during the first hour after addition of 10–9 M AVP to the cell medium, after which time it gradually increased (Fig. 2, D and E). Contrary to AQP2 protein, AQP2 mRNA expression gradually increased over the entire period after addition of 10–9 M AVP to the cell medium (Fig. 2F).
The specificity of AVP washout acting on AQP2 expression and the role played by the V2 receptor in this process were investigated. Real-time PCR analysis revealed high V2-receptor mRNA content (cycle threshold for V2 receptor was <26 and that for Po was <18; these values were not significantly altered by 24 h of 10–10 M AVP stimulation or by either 30 min of AVP chase or 30 min of 10–9 M AVP stimulation), indicating stable expression of this receptor mRNA in mpkCCDcl4 cells. Expression levels of AQP2 protein were similarly increased in cells subjected 30 min to AVP chase and cells subjected to 10–8 M SR-121463B, a nonpeptidic, competitive, and specific V2-receptor antagonist (39), whereas addition of 10–10 M AVP immediately after AVP washout blunted the increase of AQP2 protein expression compared with cells subjected for 24 h to 10–10 M AVP (Fig. 3, A and B). These results indicate that upregulated AQP2 protein expression shortly after AVP washout results from blunted V2-receptor activity and not from replacement of the incubation medium. To examine the role played by the V2 receptor in downregulated AQP2 protein expression that occurs shortly after 10–9 M AVP administration, 10–10 M AVP-pretreated cells were treated with 10–9 M dDAVP for 30 min. Both AVP and dDAVP decreased AQP2 protein expression to similar extents (Fig. 3, C and D, lanes 1–3), indicating that the V2 receptor plays a key role in this process. This was further confirmed by treating AVP-pretreated cells with 10–8 M SR-121463B for 30 min followed by an additional 30 min of incubation in the presence of 10–9 M AVP. Although 10–9 M AVP decreased AQP2 protein expression in the absence of SR-121463B, the increased expression levels of AQP2 protein induced by SR-121463B were not altered by 10–9 M AVP (Fig. 3, C and D, lanes 4–7).
Together, the time course experiments performed in 10–10 M AVP-pretreated cells subjected to AVP chase or to 10–9 M AVP suggest that V2-receptor activity mediates posttranscriptional inhibition of AQP2 protein expression in mpkCCDcl4 cells. Such a negative feedback mechanism induced by AVP would attenuate expression of AQP2 protein.
AVP enhances AQP2 protein degradation in mpkCCDcl4 cells. An AVP-induced mobilization of sarcoplasmic/endoplasmic reticulum Ca2+ stores that leads to attenuated protein synthesis during the first 30 min after AVP stimulation via the V1 receptor has been demonstrated in H9c2 ventricular myocytes (37). AVP-induced intracellular Ca2+ mobilization via stimulation of V2 receptors has also been demonstrated in perfused rat renal CD (9, 45), an event that participates in regulated AQP2 trafficking (9). Because BAPTA has been shown to block both the AVP-induced increase in intracellular free Ca2+ and the ensuing increase of osmotic water permeability (9, 45), we tested whether the decreased levels of AQP2 content shortly after 10–9 M AVP administration may be due, at least in part, to intracellular Ca2+ mobilization. For this purpose, 10–10 M AVP-pretreated mpkCCDcl4 cells were treated with 10–5 M BAPTA-AM for 1 h prior to 30 min of 10–9 M AVP stimulation. The extent of the decrease of AQP2 protein content was similar between cells treated or not with BAPTA-AM (Fig. 4, A and B, compare decreased expression between lanes 1 and 2 and between lanes 3 and 4), indicating that decreased AQP2 protein expression following enhanced AVP stimulation is not due to intracellular Ca2+ mobilization.
We further examined the role that altered translational activity may play in the increased or decreased expression levels of AQP2 protein that occur shortly after AVP chase and 10–9 AVP administration, respectively. We investigated altered AQP2 mRNA translation by treating 10–10 M AVP-pretreated mpkCCDcl4 cells for 24 h with 2 x 10–5 M of the translational inhibitor cycloheximide for 30 min before 30 min of AVP chase or 10–9 M AVP stimulation. Cells treated with cycloheximide expressed significantly less AQP2 protein than cells treated with 10–10 M AVP alone (Fig. 4, C and D, compare lanes 4 and 1), reflecting reduced synthesis of AQP2 protein. Importantly, increased AQP2 protein content induced by 30 min of AVP chase was not different between cells treated or not with cycloheximide (Fig. 4, C and D, compare lanes 2 and 5). Moreover, the extent of decreased AQP2 protein content induced by 30 min of 10–9 M AVP stimulation was similar between cells treated or not with cycloheximide (Fig. 4, C and D, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). These results strongly suggest that AQP2 mRNA translation in mpkCCDcl4 cells is not altered immediately after altered V2-receptor activity.
We have previously shown that both lysosomal and proteasomal pathways participate in AQP2 protein degradation in mpkCCDcl4 cells (14). We examined AQP2 protein degradation shortly following AVP removal or AVP supplementation by treating 10–10 M AVP-pretreated cells with a lysomal or proteasomal inhibitor for 8 h, after which period AVP was washed from the medium or readministered at a concentration of 10–9 M for an additional 30 min. Chloroquine (10–4 M), a weak base that increases lysosomal pH thereby inhibiting the proteolytic activity of lysosomal enzymes, increased AQP2 protein content compared with untreated cells (Fig. 5, A and B, compare lanes 4 and 1). Results show that the increase of AQP2 protein content induced after 30 min of AVP chase was similar between cells treated or not with chloroquine (Fig. 5, A and B, compare lanes 2 and 5). In addition, the decrease in AQP2 protein content induced after 30 min by 10–9 M AVP was attenuated in cells treated with chloroquine (Fig. 5, A and B, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). Administration of 5 x 10–6 M lactacystin, a potent and specific inhibitor of the proteasomal pathway, produced results similar to chloroquine. Lactacystin increased AQP2 protein content compared with untreated cells (Fig. 5, C and D, compare lanes 4 and 1). Results show that the extent of increased AQP2 protein expression induced after 30 min of AVP chase was similar between cells treated or not with lactacystin (Fig. 5, C and D, compare lanes 2 and 5). In addition, lactacytin attenuated decreased AQP2 protein expression induced after 30 min by 10–9 M AVP (Fig. 5, C and D, compare decreased expression between lanes 1 and 3 and between lanes 4 and 6). Together, our results reveal a transient decrease of AQP2 protein degradation shortly following abrogated V2-receptor activity and increased AQP2 degradation shortly following enhanced V2-receptor stimulation. In addition, our results suggest that both lysosomal and proteasomal pathways participate in this process.
The intracellular mechanisms that participate in the transient alterations of AQP2 protein content following altered V2-receptor activity was further investigated in mpkCCDcl4 cells. Modulation of renal water transport by PGE2 is well established (13) and at least partly results from AVP antagonism. PGE2 was additionally shown to reverse AVP-induced AQP2 translocation to the plasma membrane (46) and inhibition of cyclooxygenase-2 (COX-2), a key player in PGE2 synthesis, prevented downregulated AQP2 expression induced by bilateral urelateral obstruction (8, 31). Because COX-2 expression is induced by AVP in the inner medulla (47), we investigated whether the transient alterations of AQP2 protein expression after AVP removal or supplementation are linked to reduced or enhanced PGE2 synthesis. This was performed by comparing altered AQP2 protein expression between cells treated or not with 10–5 M indomethacin, a classical COX inhibitor. As shown in Fig. 6, indomethacin did not significantly alter the transient increase or decrease of AQP2 protein content, indicating that altered PGE2 synthesis is not responsible for this event.
We next investigated the role of PKA activity in AVP-inducible AQP2 posttranscriptional processing by comparing altered AQP2 protein expression between cells treated or not with 5 x 10–6 M myristoylated PKA inhibitor (PKI; Calbiochem). The efficacy of PKI in mpkCCDcl4 cells was previously confirmed (16). One hour of PKI treatment significantly increased AQP2 protein abundance (Fig. 7, A and B, compare lanes 4 and 1), blunted AQP2 protein upregulation induced by AVP washout (Fig. 7, A and B, compare increased expression between lanes 1 and 2 and between lanes 4 and 5), and attenuated AQP2 protein downregulation induced by AVP supplementation (Fig. 7, A and B, compare reduced expression between lanes 1 and 3 and between lanes 4 and 6). These results strongly suggest that the transient changes of AQP2 protein abundance, and therefore degradation, resulting from altered V2-receptor activity in mpkCCDcl4 cells are largely mediated by changes in PKA activity. This incited us to investigate whether altered V2-receptor activity influences phosphorylation of AQP2 itself since AQP2 phosphorylation, at Ser-256, is mediated by PKA (20). For this purpose, we performed Western blot analysis using antiserum against AQP2 amino acids 253–262 containing phosphorylated Ser-256 to measure pAQP2 levels after 30 min of AVP chase or AVP supplementation in AVP-pretreated cells. Similar to total AQP2, i.e., the AQP2 signal obtained by the polyclonal anti-AQP2 antibody (44) (Fig. 7, C and D), the intensity of the signal corresponding to pAQP2, revealed by several distinct bands 29 kDa in size and a more diffuse band 45 kDa in size, increased in an AVP dose- and time-dependent manner (Fig. 7, E and F, compare lanes 6, 3, and 1 and compare lanes 1 and 2). Interestingly, although 30 min of AVP chase increased total AQP2 and pAQP2 abundance proportionally (Fig. 7, E and F, compare lanes 3 and 4), 30 min of AVP supplementation increased pAQP2 content (Fig. 7, E and F, compare lanes 3 and 5) despite decreased abundance of total AQP2 (Fig. 7, C and D, compare lanes 4 and 6). This observation indicates that AQP2 phosphorylation at Ser-256 may precede AQP2 degradation induced by AVP. Close inspection of pAQP2 immunoblots revealed two bands corresponding to 28 and 29 kDa, discernable in the presence of AVP. These two bands were replaced by a single band of 28 kDa in response to 30 min of AVP chase. On the other hand, 30 min of 10–9 M AVP supplementation resulted in a shift toward the 29-kDa band. We hypothesize that the 29-kDa band may result from posttranslational processing and may represent an AVP-induced AQP2 predegradation product.
DISCUSSION
We have previously shown that stimulation of mpkCCDcl4 cells with AVP induces upregulated expression of both AQP2 mRNA and protein, whereas removal of AVP from the cell medium ultimately leads to their degradation (14). In the present study, AVP washout was found to transiently increase AQP2 protein expression, whereas enhanced V2-receptor activity mirrored this effect by transiently decreasing AQP2 protein expression. The altered expression levels of AQP2 protein were not associated with altered expression of AQP2 mRNA, indicating that in addition to transcriptional control AQP2 protein expression is also regulated at a posttranscriptional level in mpkCCDcl4 cells. Posttranscriptional processing of AQP2 protein was found to depend on AQP2 protein degradation rather than altered protein synthesis, a process involving both lysosomal and proteasomal degradation pathways. Together, our results suggest that in cultured mpkCCDcl4 cells AVP induces both AQP2 gene transcription and enhances AQP2 protein degradation. The physiological context of AVP-mediated increased degradation of AQP2 protein shown in the present study may be comparable to that of investigations that demonstrate conditional protein degradation of specific targets induced by specific stimuli. For instance, lysosomal degradation of E-cadherin induced by v-Src was found to participate in cell-to-cell dissociation during epithelial to mesenchymal transition (34), and proteasome-dependent degradation of the transcription factor AP-2 by TNF- was found to play a critical role in TNF--induced apoptosis (32).
Most reports from literature indicate coordinated expression levels between AQP2 mRNA and protein. For instance, the expression levels of both AQP2 mRNA and protein increased in rats treated with AVP (17, 29) and decreased in rats treated with V2-receptor antagonists (10, 23, 26). These observations suggest that AQP2 gene transcription, and possibly selective degradation of AQP2 mRNA, plays a capital role in controlled AQP2 protein abundance. However, under certain conditions, uncoupled expression between AQP2 protein and mRNA has been described. The enhanced water permeability of isolated IMCD from rats made chronically hypercalcemic by dihydrotachysterol was accompanied by significantly reduced expression levels of AQP2 protein, but not by mRNA (38). Polyuria exhibited in rats deprived of food for 24 h was also shown to be accompanied by decreased AQP2 protein, but not mRNA, content in the cortex, whereas the outer medulla displayed decreased expression of both AQP2 protein and mRNA (1, 42). These observations are indicative of AQP2 posttranscriptional processing occurring in vivo.
Further indications of in vivo regulation of AQP2 protein content occurring at a posttranscriptional level has been provided by experiments performed with V2-receptor antagonists. Administration of OPC-31260 in rats revealed decreased AQP2 mRNA in inner medulla expression occurring within 30 min of treatment, which persisted 24 h later (10). AQP2 protein content, however, was found to be similar between OPC-31260- and sham-operated animals after 1 and 24 h of treatment and only decreased in animals administered with OPC-31260 for 60 h (10, 23). The lag of AQP2 protein degradation compared with that of AQP2 mRNA may possibly be due to slow AQP2 protein degradation occurring in these animals, as opposed to rapid degradation of AQP2 mRNA. However, rapidly downregulated expression of AQP2 protein has been described under certain conditions. For instance, rats fed a K+-free diet displayed decreased cortical AQP2 mRNA and protein abundance after 12 h of K+ deprivation (2). Downregulated expression of both AQP2 mRNA and protein was also observed after 24 h in inner medulla of obstructed kidneys (12). Because both of these conditions do not arise from decreased plasma AVP concentration (12, 25, 43), it is possible that reduced V2-receptor activity in OPC-31260-treated animals temporally reduces AQP2 protein degradation, an event that occurs concurrently with decreased AQP2 mRNA expression. This effect would be similar to that found in the present study produced in cultured mpkCCDcl4 cells in which AVP chase or SR-121463B administration transiently increased AQP2 protein, but not mRNA, abundance via decreased AQP2 protein degradation.
Interestingly, the decreased abundance of AQP2 protein, but not mRNA, in both dihydrotachysterol-treated and fasted animals occurred independently of changes in AVP plasma concentration (36, 42). Similar changes in AQP2 expression resulting from fasting were also observed in AVP-deficient Brattleboro rats (42). Together with the results of the present study, these observations indicate that posttranscriptional changes in AQP2 protein content may be induced by AVP and/or by factors acting independently of AVP, possibly by acting via pathways normally stimulated by AVP and that influence protein degradation. Although recent studies have demonstrated decreased AQP2 abundance resulting from increased COX activity (8, 31), our results indicate that, in mpkCCDcl4 cells, this pathway is not responsible for altered AQP2 protein abundance occurring in response to acute variations of V2-receptor activity.
The results of the present study suggest that PKA controls AQP2 abundance at two distinct levels: it promotes AQP2 mRNA expression on the one hand and retroactively constrains AQP2 protein abundance on the other hand, most likely by promoting AQP2 degradation. The rapid (30 min) effects produced by AVP washout and AVP supplementation on AQP2 protein abundance additionally suggest that the mechanism responsible for these effects consists of posttranscriptional processing, i.e., phosphorylation, as opposed to transcriptional-mediated processing. This is further supported by the observation that both effects occur regardless of the presence or absence of the translational inhibitor cycloheximide. The observation that pAQP2 abundance is increased after 30 min of AVP supplementation indicates that AQP2 degradation is preceded by AQP2 phosphorylation at Ser-256. AQP2 phosphorylation is necessary for its cell surface expression (19). The observation made by the present study that links PKA activity to AVP-induced AQP2 protein degradation, possibly via AQP2 phosphorylation, in mpkCCDcl4 cells may be tentatively compared with observations made in renal CD8 cells and IMCD cells, establishing the necessity of active PKA for AQP2 cell surface expression, a process that involves phosphorylation of AQP2 itself and of other proteins that mediate AQP2 exocytosis, such as RhoA (27, 40). Although further investigation is needed, these observations indicate that AQP2 cell surface expression may be tightly regulated by PKA via dual stimulation of AQP2 exocytosis and degradation.
In conclusion, our results indicate that AQP2 protein abundance is regulated at a posttranscriptional level via increased AQP2 protein degradation induced by enhanced V2-receptor activity. This process is dependent on PKA activity and involves phosphorylation of AQP2 itself. PKA may therefore play a dual role in controlling AQP2 abundance: it increases AQP2 gene transcription following V2-receptor stimulation and retroactively controls AQP2 protein content by promoting its degradation.
GRANTS
This work was supported by the Swiss National Foundation for Science Grant 3100-067878.02, by a grant from the Carlos et Elsie De Reuter Foundation to E. Feraille, and by a grant from the Novartis Foundation to U. Hasler.
FOOTNOTES
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.
E. Feraille and P.-Y. Martin contributed equally to this work.
REFERENCES
Amlal H, Chen Q, Habo K, Wang Z, and Soleimani M. Fasting downregulates renal water channel AQP2 and causes polyuria. Am J Physiol Renal Physiol 280: F513–F523, 2001.
Amlal H, Krane CM, Chen Q, and Soleimani M. Early polyuria and urinary concentrating defect in potassium deprivation. Am J Physiol Renal Physiol 279: F655–F663, 2000.
Asahina Y, Izumi N, Enomoto N, Sasaki S, Fushimi K, Marumo F, and Sato C. Increased gene expression of water channel in cirrhotic rat kidneys. Hepatology 21: 169–173, 1995.
Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, and Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol 10: 923–934, 1999.
Bustamante M, Hasler U, Kotova O, Chibalin AV, Mordasini D, Rousselot M, Vandewalle A, Martin PY, and Feraille E. Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells. Am J Physiol Renal Physiol 288: F334–F344, 2005.
Carranza ML, Feraille E, and Favre H. Protein kinase C-dependent phosphorylation of Na+-K+-ATPase -subunit in rat kidney cortical tubules. Am J Physiol Cell Physiol 271: C136–C143, 1996.
Chabardes D, Firsov D, Aarab L, Clabecq A, Bellanger AC, Siaume-Perez S, and Elalouf JM. Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content. J Biol Chem 271: 19264–19271, 1996.
Cheng X, Zhang H, Lee HL, and Park JM. Cyclooxygenase-2 inhibitor preserves medullary aquaporin-2 expression and prevents polyuria after ureteral obstruction. J Urol 172: 2387–2390, 2004.
Chou CL, Yip KP, Michea L, Kador K, Ferraris JD, Wade JB, and Knepper MA. Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin. J Biol Chem 275: 36839–36846, 2000.
Christensen BM, Marples D, Jensen UB, Frokiaer J, Sheikh-Hamad D, Knepper M, and Nielsen S. Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution. Am J Physiol Renal Physiol 275: F285–F297, 1998.
Ecelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, and Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 1852–1863, 1997.
Frokiaer J, Christensen BM, Marples D, Djurhuus JC, Jensen UB, Knepper MA, and Nielsen S. Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction. Am J Physiol Renal Physiol 273: F213–F223, 1997.
Harris RC and Breyer MD. Physiological regulation of cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol 281: F1–F11, 2001.
Hasler U, Mordasini D, Bens M, Bianchi M, Cluzeaud F, Rousselot M, Vandewalle A, Feraille E, and Martin PY. Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells. J Biol Chem 277: 10379–10386, 2002.
Hasler U, Mordasini D, Bianchi M, Vandewalle A, Feraille E, and Martin PY. Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells. J Biol Chem 278: 21639–21648, 2003.
Hasler U, Vinciguerra M, Vandewalle A, Martin PY, and Feraille E. Dual effects of hypertonicity on aquaporin-2 expression in cultured renal collecting duct principal cells. J Am Soc Nephrol 16: 1571–1582, 2005.
Hayashi M, Sasaki S, Tsuganezawa H, Monkawa T, Kitajima W, Konishi K, Fushimi K, Marumo F, and Saruta T. Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney. J Clin Invest 94: 1778–1783, 1994.
Hayashi M, Sasaki S, Tsuganezawa H, Monkawa T, Kitajima W, Konishi K, Fushimi K, Marumo F, and Saruta T. Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney. J Clin Invest 94: 1778–1783, 1994.
Kamsteeg EJ, Heijnen I, van Os CH, and Deen PM. The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers. J Cell Biol 151: 919–930, 2000.
Kuwahara M, Fushimi K, Terada Y, Bai L, Marumo F, and Sasaki S. cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J Biol Chem 270: 10384–10387, 1995.
Laursen UH, Pihakaski-Maunsbach K, Kwon TH, Ostergaard Jensen E, Nielsen S, and Maunsbach AB. Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus. Nephron Exp Nephrol 97: 1–16, 2004.
Ma T, Hasegawa H, Skach WR, Frigeri A, and Verkman AS. Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. Am J Physiol Cell Physiol 266: C189–C197, 1994.
Marples D, Christensen BM, Frokiaer J, Knepper MA, and Nielsen S. Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats. Am J Physiol Renal Physiol 275: F400–F409, 1998.
Marples D, Christensen S, Christensen EI, Ottosen PD, and Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 95: 1838–1845, 1995.
Marples D, Frokiaer J, Dorup J, Knepper MA, and Nielsen S. Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. J Clin Invest 97: 1960–1968, 1996.
Murillo-Carretero MI, Ilundain AA, and Echevarria M. Regulation of aquaporin mRNA expression in rat kidney by water intake. J Am Soc Nephrol 10: 696–703, 1999.
Nejsum LN, Zelenina M, Aperia A, Frokiaer J, and Nielsen S. Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation. Am J Physiol Renal Physiol 288: F930–F938, 2005.
Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, and 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.
Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663–11667, 1993.
Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, and Knepper MA. Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205–244, 2002.
Norregaard R, Jensen BL, Li C, Wang W, Knepper MA, Nielsen S, and Frokiaer J. COX-2 inhibition prevents downregulation of key renal water and sodium transport proteins in response to bilateral ureteral obstruction. Am J Physiol Renal Physiol 289: F322–F333, 2005.
Nyormoi O, Wang Z, Doan D, Ruiz M, McConkey D, and Bar-Eli M. Transcription factor AP-2 is preferentially cleaved by caspase 6 and degraded by proteasome during tumor necrosis factor -induced apoptosis in breast cancer cells. Mol Cell Biol 21: 4856–4867, 2001.
Ohara M, Martin PY, Xu DL, St. John J, Pattison TA, Kim JK, and Schrier RW. Upregulation of aquaporin 2 water channel expression in pregnant rats. J Clin Invest 101: 1076–1083, 1998.
Palacios F, Tushir JS, Fujita Y, and D’Souza-Schorey C. Lysosomal targeting of E-cadherin: a unique mechanism for the down-regulation of cell-cell adhesion during epithelial to mesenchymal transitions. Mol Cell Biol 25: 389–402, 2005.
Promeneur D, Kwon TH, Frokiaer J, Knepper MA, and Nielsen S. Vasopressin V2-receptor-dependent regulation of AQP2 expression in Brattleboro rats. Am J Physiol Renal Physiol 279: F370–F382, 2000.
Puliyanda DP, Ward DT, Baum MA, Hammond TG, and Harris HW Jr. Calpain-mediated AQP2 proteolysis in inner medullary collecting duct. Biochem Biophys Res Commun 303: 52–58, 2003.
Reilly BA, Brostrom MA, and Brostrom CO. Regulation of protein synthesis in ventricular myocytes by vasopressin. The role of sarcoplasmic/endoplasmic reticulum Ca2+ stores. J Biol Chem 273: 3747–3755, 1998.
Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, and Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978–F985, 1998.
Serradeil-Le Gal C, Lacour C, Valette G, Garcia G, Foulon L, Galindo G, Bankir L, Pouzet B, Guillon G, Barberis C, Chicot D, Jard S, Vilain P, Garcia C, Marty E, Raufaste D, Brossard G, Nisato D, Maffrand JP, and Le Fur G. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729–2738, 1996.
Tamma G, Klussmann E, Maric K, Aktories K, Svelto M, Rosenthal W, and Valenti G. Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells. Am J Physiol Renal Physiol 281: F1092–F1101, 2001.
Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414–F422, 1996.
Wilke C, Sheriff S, Soleimani M, and Amlal H. Vasopressin-independent regulation of collecting duct aquaporin-2 in food deprivation. Kidney Int 67: 201–216, 2005.
Wilson DR. Pathophysiology of obstructive nephropathy. Kidney Int 18: 281–292, 1980.
Xu DL, Martin PY, Ohara M, St John J, Pattison T, Meng X, Morris K, Kim JK, and Schrier RW. Upregulation of aquaporin-2 water channel expression in chronic heart failure rat. J Clin Invest 99: 1500–1505, 1997.
Yip KP. Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct. J Physiol 538: 891–899, 2002.
Zelenina M, Christensen BM, Palmer J, Nairn AC, Nielsen S, and Aperia A. Prostaglandin E2 interaction with AVP: effects on AQP2 phosphorylation and distribution. Am J Physiol Renal Physiol 278: F388–F394, 2000.
Zhang MZ, Sanchez Lopez P, McKanna JA, and Harris RC. Regulation of cyclooxygenase expression by vasopressin in rat renal medulla. Endocrinology 145: 1402–1409, 2004.(Udo Hasler, Sren Nielsen, Eric Feraille,)