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【摘要】 The -subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either -subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed -subunit protein under isotonic conditions. Clones expressing the b isoform showed enhanced survival at lethal acute hypertonicity compared with either a isoform or empty vector (control) expressing clones. We also evaluated the loss of -subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable -subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of mRNA and protein was essentially undetectable. The impact of silencing -subunit expression resulted in a 70% reduction at 48 h ( P < 0.01) in cell survival compared with empty vector (control) clones. siRNA clones showed a 45% decrease in myo -inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the -subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.
【关键词】 hypertonicity osmoregulation smallinterference RNA
THE CELLS THAT INHABIT THE hypertonic environment of the inner medulla possess a number of adaptive mechanisms that allow them to survive this harsh environment. This survival is mediated initially by the activation of ion transport systems ( 9 ) and thereafter by the cellular accumulation of a number of organic osmolytes ( 12 ). It has become increasingly evident that in addition to the proteins required for the cellular uptake and/or synthesis of these osmolytes (transporters and enzymes), hypertonic stress brings about a coordinated response involving the up- and downregulation of hundreds of genes ( 16, 17 ), many of which may be critical to cell viability and cell adaptation. Our laboratory studied the role of the - and -subunits ( 5 ) and the more recently described -subunit ( 6 ) of Na-K-ATPase on cell survival and adaptation in response to hypertonicity in inner medullary collecting duct (IMCD)3 cells. We have done so both in cultured cells and in rodents at various states of hydration ( 5, 6, 8 ). In these studies, maneuvers that decreased the expression of the -subunit were consistently associated with a decrease of cell survival in cultures exposed to hypertonic conditions. Specifically, inhibition of the osmotically stimulated JUN kinase and PI3 kinase signaling pathways results in both a drastic decrease in -subunit synthesis and in cell survival ( 6 ). Likewise, replacement of chloride (NaCl) with acetate (NaAc) impairs both the -subunit synthesis and the adaptation of IMCD3 cells to hypertonicity ( 8 ). Although these observations strongly suggest that the -subunit is crucial for the survival of IMCD3 cultures exposed to hypertonic stress ( 6 ), a direct cause and effect relationship could not be established. Therefore, the purpose of the present study was to directly examine the role of the -subunit of Na-K-ATPase in osmoadaptation by obtaining clones that either overexpress or silence the expression of the -subunit.
MATERIALS AND METHODS
Materials
Cell culture medium, serum, and antibiotics were obtained from Invitrogen (Carlsbad, CA). Antibodies to the -subunit of Na-K-ATPase (to the common COOH-terminal end and recognizing both a and b splice variants) were generously provided by Dr. S. Karlish (The Weizmann Institute of Science, Rehovot, Israel). Antibodies to the 1 subunit of Na-K-ATPase were purchased from Upstate Biotechnology (Lake Placid, NY).
Cell Culture
The established murine inner medullary collecting duct cell line IMCD3 was previously provided by Dr. S. Gullans (Boston, MA). Cell stocks were frozen in liquid N 2 and propagated in 1:1 mixture of DMEM and Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin (GIBCO, Rockville, MD). In experiments involving hypertonic stress, the media in culture dishes were exchanged for that with added NaCl to the specified osmolality depending on the experiment. Osmolality was determined with an Advanced Instruments Micro-Osmometer (model 3300, Norwood, MA).
Cell Viability Experiments
Cell viability in tissue culture stress experiments was determined by cell counts following incubation at stress. Cells were grown in 24-well flat bottom tissue culture plates (#35-3047, Falcon BD Labware, Franklin Lakes, NJ) with each experimental time point performed in triplicate. At the defined time point, medium was removed, wells were then washed vigorously with 2 ml of new media, and the media were removed and 1 ml of trypsin was added and incubated at 37°C for 10 min. An additional 1.5 ml of media were then added to each well and the cells were resuspended and a 20-µl aliquot of cell suspension was diluted 1:1 with Trypan Blue (GIBCO, Grand Island, NY). Viable cells that exclude Trypan blue were counted using a hemocytometer (Fisher, Pittsburgh, PA). Greater than 96% of the cells excluded Trypan blue and were determined viable and could be successfully replated. Data from cell counts were similar to that obtained using the Cell Titer 96 assay (MTS reagent, Promega, Madison, WI) albeit with greater reproducibility. Floating cells were 94%) and did not reattach and grow when replated in fresh media. Osmotic stress, both sublethal and lethal (550 and 675 mosmol/kgH 2 O), was provided by the addition of 5 M NaCl to growth media. In osmotic stress experiments with mannitol, powder mannitol was added to the medium to adjust the osmolality before filter sterilization and use in experiments. Lethal UV irradiation was applied using a Mineralight R-52G (UVP, San Gabriel, CA) short-wavelength UV lamp. Following irradiation, 24-well plates were incubated as normal. Thermal stress was provided by incubating 24-well plates in a CO 2 incubator (model 3154, Forma Scientific, Marietta, OH) set to control temperature at 42°C.
Western Analysis
Cell lysates were prepared from confluent cell cultures in 100 x 20-mm tissue culture dishes as previously described ( 5, 6 ). Sample protein content was determined by the BCA protein assay (Pierce, Rockford, IL). Depending on the experiment, from 25 to 150 µg of protein were loaded per lane for PAGE analysis. Gels were visualized using an alkaline phosphatase secondary antibody and Lumi-Phos reagent (Pierce) as described by the manufacturer. Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the one-dimensional Image Software (Kodak Digital Science, Rochester, NY).
Construction of Expression Vectors
The complete sequences of a and b mRNAs including both the 5' and 3' UTRs were previously submitted to GenBank ( AY626243 and AY626244, respectively). The -subunit overexpression vectors were constructed using the pIRES (Invitrogen) cloning vector as previously described ( 7 ). The pIRES vector contains the CMV promoter and constitutively expresses the insert containing the complete sequence for a or b including all of the 3' and 5' UTR sequences. The -subunit silencing vector was constructed employing the pSupressorNeo vector (Biocarta, San Diego, CA). The following primers were designed as described by the manufacturer: si F 5'-TCGAGAAGGGGACAGAGAATCCCTTCGAGTACTGGAAGGGATTCTCTGTCCCCTTTTTTT-3'; si R 5'-CTAGAAAAAAAGGGGACAGAGAATCCCTTCCAGTACTCGA, AGGGATTCTCTGTCCCCTTC-3'.
The sequence targets the mRNA between positions 49 to 69 (conserved sequence) and contains a unique Rsa I restriction site in the loop structure to facilitate the screening of positive clones. The si sequence was annealed and ligated in the Sal I/ Xba I digested vector. During this step, the Sal I restriction site was destroyed, which facilitates the screening of positive clones.
Cell Transfection
IMCD3 cultures were transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Stable transfectants (clones) were selected from colonies growing in plates from a 10-fold dilution series in media prepared with 500 µg/ml of G-418 antibiotic (RPI, Mt. Prospect, IL). Clones were subjected to a second round of 10-fold dilutions and replated in G418 media and a second colony selection was performed to provide clean clones for further analysis.
Quantitative PCR
Cytosolic RNA was isolated from confluent cultures in 100 x 20-mm tissue culture dishes as previously drescribed ( 5 ) using the RNeasy kit (Qiagen, Valencia, CA). Before quantitative PCR, RNA integrity was assessed by capillary electrophoresis using an Agilent Bioanalyzer [model 2100, Foster City, CA (using the 28S-to-18S rRNA ratio)]. RNA was converted to cDNA using the Omniscript Reverse Transcriptase kit (Qiagen) as described by the manufacturer. Quantitative PCR primers (50 nM each) were designed using Beacon Designer 4.0 software (Premier Biosoft International, Palo Alto, CA) targeting the conserved sequence of the -subunit, the unique sequences for a and b isoforms as described in Table 1.
Table 1. Specific primers used for QPCR analysis
QPCR was performed using the Quantitect SYBR Green PCR kit (Qiagen) on a Bio-Rad I-Cycler (Hercules, CA). QPCR runs were analyzed by agarose gel electrophoresis and melt curve to verify the correct amplicon was produced. To normalize the cDNA concentration in samples, QPCR assays for 18S RNA were performed using the 18s RNA Taq man kit (Applied Biosystems, Foster City, CA).
Assay of myo-Inositol-Na Cotransporter
Myo-inositol uptake was measured as previously described ( 18, 20 ) with the following modifications. After incubation with the radioactive compounds ( myo -inositol or methoxyinulin), the medium was saved to measure their concentrations, the cells were lysed in 1% SDS, and the lysates were sonicated in a Vibra Cell (Sonic & Materials, Danbury, CT) to fragment the DNA and reduce viscosity; aliquots of each reaction were used to measure radioactivity and proteins. The calculations to correct for trapping and/or nonspecific binding by using inulin as a nonpenetrator standard were done as previously described ( 4 ).
Statistics
Results were analyzed by ANOVA and Tukey-Kramer multiple comparisons test using the InStat software package (GraphPad software, San Diego, CA). A value of P < 0.05 is considered significant.
RESULTS
Overexpression of Na-K-ATPase -Subunit
Multiple independent a and b overexpressing clones were obtained by transfection of IMCD3 cells with a pIRES expression vector containing the complete sequence of the -subunit mRNA including the CDS and the regulatory 5' and 3' UTRs. Clones were selected in G418-containing media and quantified for message by QPCR and for protein by Western blot under isotonic conditions.
Effects on mRNA and protein levels. As shown in Table 2, IMCD3 cells have very low amounts of -subunit message under isotonic conditions. In contrast, transfected cells expressed four orders of magnitude greater levels of -subunit mRNA under isotonic conditions. This level of transcription is at least two orders of magnitude greater than in IMCD3 cells exposed to NaCl hyperosmotic stress (550 mosmol/kgH 2 O, 48 h). However, we demonstrated that the -subunit is regulated at both the transcriptional and translational level and that message levels do not correlate with protein levels under nonstress conditions ( 7, 14 ). The expression of -subunit protein in overexpressing clones is depicted in Fig. 1. All transfected clones express substantial amounts of -subunit protein under isotonic conditions although at a level much lower than in cells exposed to either acute or chronic hypertonicity. Alternatively, -subunit protein is not detected under such conditions in either IMCD3 cells or in cells transfected with the empty vector. It is noteworthy that all b clones produce about one order of magnitude more protein than all a clones despite the fact that they produce very similar amounts of mRNA (as shown in Table 2 ).
Table 2. Overexpression of -subunit isoforms in clones transfected with the stable pIRES vector
Fig. 1. Stable -subunit-expressing clones were obtained by transfecting inner medullary collecting duct (IMCD)3 cells with a pIRES vector containing the complete sequence of either a or b and selection with the antibiotic G418 compared with the empty vector controls. Western blot analysis of multiple clones shows that individual clones produce the corresponding variant of the -subunit under isotonic conditions. Two-hundred micrograms of protein were loaded per well and data demonstrate that b clones produce substantially more protein than a clones.
Effect on cell survival in response to lethal hypertonic stress. We ( 5, 6 ) and others ( 2 ) previously demonstrated that cultures of IMCD3 cells do not withstand acute osmotic stress that exceed 550 mosmol/kgH 2 O. To assess whether the presence of the -subunit confers IMCD3 cells the ability to withstand lethal hypertonic stress, we subjected independent a and b overexpressing clones to media adjusted to 675 mosmol/kgH 2 O for increasing times. As shown in Fig. 2, b overexpressing clones demonstrate on average a 79% survival at 24 h and a 77% survival even after 48 h of incubation. Suprisingly, IMCD3 cells overexpressing a demonstrate a rapid loss of cell viability over the same time period. This loss in viability is similar to that for control IMCD3 cells (empty vector), that had 15 and 6% survival at 24 and 48 h, respectively.
Fig. 2. Effect of lethal osmotic stress (675 mosmol/kgH 2 O) on IMCD3 cell survival of independent, stable Na-K-ATPase -subunit-expressing clones. Data represent means ± SE of 4 independent clones (3 experiments, n = 12) and depict the percentage of surviving cells with respect to time 0 cultures. Differences between a ( ) and empty vector clones ( ) were not significant ( P 0.1). Differences between b clones ( ) and either a or empty vector clones were significant ( P < 0.002 at 24 h and P < 0.001 at 48 h).
Effect of -subunit overexpression on IMCD3 cell survival with various stresses. To probe the specificity of the protection conferred by the overexpression of b, we subjected the representative clone, b4, and the empty vector control cells to three different types of stresses. As depicted in Fig. 3, the b4 overexpressing clone did not demonstrate improved cell survival compared with control cells when subjected to a nonionic hyperosmotic stress using mannitol ( Fig. 3 A ), UV irradiation ( Fig. 3 B ), and thermal exposure ( Fig. 3 C ).
Fig. 3. Effect of various stresses on the survival of -subunit-overexpressing clones. A : effect of an acute lethal osmotic stress employing the nonionic osmolyte, mannitol (675 mosmol/kgH 2 O). Both the representative b4 clone and the empty vector control cells demonstrated a similar loss in viability. B : effect of lethal UV irradiation (192 J/m 2 ) on cell viability for the b4 clone compared with the empty vector controls demonstrating a comparable loss in cell survival. C : effect of lethal thermal stress (42°C) on the viability of b4 clones compared with the empty vector controls indicating a similar loss in cell survival. All experiments were performed in triplicate and data represent means ± SE from 3 independent experiments.
Silencing of Na-K-ATPase -Subunit
Multiple independent -subunit silencer-expressing clones were obtained by transfection of IMCD3 cells with a pSupressor expression vector containing the -specific silencer RNA sequence. Clones were selected in G418-containing media and quantified for reduced message and protein under sublethal hypertonic conditions (550 mosmol/kgH 2 O, 48 h).
Effects on mRNA and protein levels. Clones constitutively expressing siRNA were subjected to sublethal hypertonic stress, and -subunit message levels were measured by QPCR as summarized in Table 3. Exposure of empty vector IMCD3 cells to 550 mosmol/kgH 2 O for 48 h results in a 60-fold increment in message, while in siRNA clones, this same stress produces a less than fourfold increase. This strong inhibition of -subunit message is reflected in protein expression as shown in Fig. 4. Of five independent clones evaluated by Western blot, four demonstrated levels of protein expression that are below detection. One clone (#4) contained roughly 20% of empty vector (control) -subunit protein expression. As a comparison, all clones contained comparable levels of the 1 -subunit of Na-K-ATPase following acute osmotic stress demonstrating that the -silencing effect is very specific.
Table 3. Effect of stable -subunit siRNA vector on -subunit mRNA copy number during osmotic stress of IMCD3 cells
Fig. 4. Western blot analysis of 5 independent clones of IMCD3 transfected with a stable siRNA-generating vector following a 48-h sublethal osmotic stress. Most siRNA clones demonstrated complete suppression of protein while clone #4 was determined to express 20% of the protein expressed in empty vector controls. Data shown are a representative Western blot from 3 independent determinations with 200 µg of protein loaded per lane. Data also indicate no effect on the typical upregulation of the 1 -subunit of Na-K-ATPase.
Effect on cell survival in response to sublethal hypertonic stress. To evaluate the physiological function of the failure to generate the -subunit in the osmotic stress response, we assessed the degree of cell viability of our clones when exposed to what is normally a level of osmotic stress that is well tolerated, namely 550 mosmol/kgH 2 O for 24 and 48 h. These data are depicted in Fig. 5 80% survival. In contrast, silenced clones consistently displayed reduced survival ability, at both 24 h survival was 40% ( P < 0.04) and at 48 h survival was 30% ( P < 0.01) compared with the initial cell numbers. Evaluation of siRNA clone #4 which produced a modest amount of -subunit protein demonstrated an intermediate level of cell survival.
Fig. 5. Effect of a sublethal osmotic stress (550 mosmol/kgH 2 O) on cell survival of IMCD3 clones transfected with a stable siRNA vector. Data represent means ± SE for the percentage of surviving cells with respect to time 0. Data shown for the siRNA clones ( ) were from 4 independent clones (3 experiments, n = 12) and were significantly different from the empty vector controls ( ), P < 0.04 at 24 h and P < 0.02 at 48 h. Data for the partial -subunit-expressing clone, siRNA #4 ( ), demonstrate an intermediate level of survival.
Effect on myo-inositol transport. The upregulation of osmolyte transporters, myo -inositol among them, is a well-known response of renal cells to increments in tonicity ( 3, 12 ). We further investigated whether the failure of -silenced cells to survive under sublethal stress was associated with alterations in the transport of inositol. As shown in Fig. 6, a 46% decrease in the Na + / myo -inositol cotransporter activity ( P < 0.005) was detected when siRNA clones were exposed to 550 mosmol/kgH 2 O for 18 h.
Fig. 6. Effect of a sublethal osmotic stress (550 mosmol/kgH 2 O for 18 h) on sodium myo -inositol cotransporter (SMIT) activity in a stable representative si-RNA clone compared with the empty vector control. Data represent means ± SE of 4 independent transport rate measurements. A 46% decrease in inositol transport activity is evident ( P < 0.005).
DISCUSSION
The cellular responses that allow kidney cells to maintain viability in the hypertonic environment of the inner medulla, and thereby permit the concentrating mechanism to function normally, have been the subject of considerable interest in various laboratories. The interest of our laboratory has focused on the -subunit of Na-K-ATPase. This protein has received considerable recent attention from the biochemical point of view and has been demonstrated to directly affect the affinity of the Na-K-ATPase pump for sodium ( 11, 13 ). The -subunit is almost exclusively localized in the kidney ( 1, 10, 15 ). However, in cell cultures of IMCD3 cells, it is undetectable under isotonic conditions. The -subunit protein is readily upregulated in response to acute increments in tonicity and is robustly expressed in cells adapted to live at 600 or 900 mosmol/kgH 2 O ( 6 ). The significance of this upregulation to the adaptive process was suggested in our previous experiments. Thus maneuvers that diminished the upregulation of the -subunit, such as inhibition of JUN kinase activity and inhibition of PI3 kinase ( 6 ), were associated with decrements in cell viability under sublethal hypertonic stress. Likewise, when hypertonicity-induced synthesis of the -subunit was altered by the replacement of the key anion chloride for another anion, survival was also diminished ( 8 ). Although these observations were highly suggestive of a critical role for the -subunit in osmoadaptation, they consistently relied on pharmacological agents that are known to have multiple effects. To supply direct support for the crucial and direct role of the -subunit in survival and adaptation of IMCD3 cells to hypertonic stress, our approach consisted of the manipulation of the amount of -subunit the cells expressed either by increasing protein expression (overexpression) or reducing protein expression (siRNA).
Our experiments in cells transfected with the full-length mRNA including the regulatory sequences for a and b isoforms yielded very interesting results. First, all clones, independent of the isoform, contained similar levels of mRNA. Second, overexpressing vector constructs allowed IMCD3 cells to produce the -subunit protein under isotonic conditions. Third, in all the analyzed clones, those tranfected with the b construct contained about one order of magnitude greater protein than clones transfected with the a isoform (see Fig. 1 ). This asymmetry in protein expression is very similar to the results that we obtained with IMCD3 cells chronically adapted to 600 and 900 mosmol/kgH 2 O ( 6 ). Taken together, these results reinforce our conjecture that there is a posttranscriptional level of regulation since the CMV promoter of the vector is unregulated ( 7 ). In this regard, IMCD3 cells may differ from other renal cells that have been described to preferentially express a in response to hypertonicity ( 19 ), an observation that may assist in explaining the cell viability results. In our experiments involving lethal hypertonic stress, clones expressing the b but not the a isoform demonstrated a 79% cell survival compared with only 15% for control cells after 24 h. This failure to observe protection with the a clones is compatible with the observation that induction of a correlates with a reduction in cell growth ( 19 ). It is thus attractive to postulate that the two isoforms impact Na-K-ATPase activity differently. Further characterization of the protection afforded IMCD3 cells upon transfection with a b overexpressing vector indicates that this effect appears to be limited to ionic hypertonic stresses (see Fig. 3 ). Given the importance of the Na-K-ATPase in the cellular distribution of ions and the central role of chloride in -subunit synthesis ( 8 ), this observation is not entirely unexpected.
Complementary experiments that inhibited the expression of the -subunit by using RNA-silencing techniques avoided the potential problems with pharmacological agents and produced results that are congruent with those described above. When silenced for the -subunit, IMCD3 clones become sensitive to sublethal osmotic stress. It should be noted that unfortunately it is impossible to create si isoform-specific clones. In addition, clones that were only partially silenced for the -subunit demonstrated an intermediate level of sensitivity to osmotic stress ( Fig. 5 ). Because the siRNA technique is very specific (i.e., no alteration in the 1 -subunit upregulation), this dramatic shift in osmotolerance confirms unambiguously the important role of the -subunit. Our results would appear to differ from those reported by Wetzel et al. ( 19 ) in which the knockdown of the -subunit abolishes cell growth delay. Important differences must be noted. Those investigators employed a transient transfection with unspecified transfection efficiency while in the present study we developed stable clones. We studied specifically inner medullary cells (IMCD3) rather than other renal epithelial cells (NRK-52E). We previously showed that IMCD3 cells are more osmotolerant than other renal cells (M1, Ref. 5 ), a fact that may relate precisely to their preferential expression of b; thus when such cells are "silenced," the consequences of downregulation of b take preponderance resulting in increased sensitivity to osmotic stress.
The mechanism whereby the -subunit confers enhanced osmotic tolerance remains to be elucidated. In this regard, we examined the effect of silencing the -subunit on the transport activity of myo -inositol, an extremely important organic osmolyte in the osmo-adaptative process. A twofold reduction in the myo -inositol transport activity was determined for clones lacking the ability to synthesize the -subunit upon sublethal osmotic stress. Since the transport of myo -inositol and most related organic osmolytes is coupled with sodium transport, silencing of the -subunit may directly affect the transmembrane sodium gradient and render transporters less effective. Direct measurement of cellular sodium and other electrolytes will be necessary to confirm or refute this hypothesis.
In summary, the present experiments represent the first time that a direct link has been demonstrated between cellular content of the -subunit of Na-K-ATPase (both increased and decreased) and the ability of renal cells to survive and adapt to ionic hypertonic stress. While these data clearly demonstrate the importance of the -subunit in osmotollerance in IMCD3 cells, they do not provide a mechanism for its effect. A growing number of studies indicate a role for FXYD proteins in modifying the affinity of Na-K-ATPase which could lead to important changes in intracellular ion and ATP levels thereby explaining the apparent reduced transport efficiency of SMIT in -silenced clones under acute hypertonic stress. At this time, the mechanism remains speculative and will require measurements of intracellular ions as well as ATP content that are not in the scope of the present investigation but are the subject of ongoing research efforts in our laboratory.
GRANTS
This work was supported by National Institutes of Health Grants DK-19928 and DK-66544 to T. Berl.
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作者单位：Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, School of Medicine, Denver, Colorado