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【摘要】 In tests of osmotic tolerance of renal inner medullary cells in tissue culture, osmolality has usually been increased in a single step, whereas in vivo the increase occurs gradually over several hours. We previously found that more passage 2 mouse inner medullary epithelial (p2mIME) cells survive a linear increase in NaCl and urea from 640 to 1,640 mosmol/kgH 2 O over 20 h (which is similar to the change that may occur in vivo) than they do a step increase. The present studies examine accompanying differences in gene expression. Among mRNAs of genes known to be protective, tonicity-responsive enhancer binding protein and aldose reductase increase with a linear but decrease with a step increase; betaine transporter BGT1 decreases with a step but not a linear increase; heat shock protein 70.1 ( HSP70.1 ) and HSP70.3 increase more with a linear than a step increase; and osmotic stress protein 94 and heme oxygenase-1 increase with a linear but decrease with a step increase. mRNAs for known urea-responsive proteins, GADD153 and Egr-1, increase with both a step and linear increase. A step increase in urea alone reduces mRNAs, similar to the combination of NaCl and urea, but a step increase in NaCl alone does not. HSP70 protein increases substantially with a linear rise in osmolality but does not change significantly with a step rise. We speculate that poorer survival of p2mIME cells with a step than with linear increase in NaCl and urea is accounted for, at least in part, by urea-induced suppression of protective genes, particularly HSP70.
【关键词】 osmotic stress organic osmolyte heat shock protein mRNA abundance
CELLS IN THE RENAL INNER MEDULLA are exposed to high interstitial osmolality that varies with urinary concentration. The lowest normal level, which occurs during water diuresis, is 600 mosmol/kgH 2 O, sodium salts being the principal solutes ( 1 ). The action of antidiuretic hormone increases it to 1,700 mosmol/kgH 2 O after several hours. Cell cultures of renal medullary cells have been used for many studies of the mechanisms by which they endure such extreme conditions. Most immortalized cells, which are customarily grown at 300 mosmol/kgH 2 O, are killed by levels of NaCl and/or urea far lower than occur in renal inner medullas in vivo. That includes mouse inner medullary collecting duct cells (mIMCD3) ( 29 ). In contrast to their survival in vivo, most mIMCD3 cells are killed by acutely raising osmolality above 600 mosmol/kgH 2 O by adding NaCl or urea or above 1,000 mosmol/kgH 2 O by adding both NaCl and urea ( 18, 30 ). Part of the difference is explained by the continuous rapid replication of immortalized mIMCD3 cells. High NaCl causes DNA double-strand breaks ( 14 ), associated with apoptosis during the S-phase of the cell cycle, in which DNA replication occurs ( 7 ). Many mIMCD3 cells are always in S because they continue to proliferate rapidly even when they are confluent ( 44 ). In contrast, inner medullary cells rarely proliferate in vivo ( 44 ). In this respect, passage 2 mouse inner medullary epithelial (p2mIME) cells more closely resemble the cells in vivo. They become contact inhibited when confluent, and far fewer are in S than are mIMCD3 cells ( 44 ). The p2mIME cells are routinely grown at 640 mosmol/kgH 2 O, similar to the osmolality in inner medullas during water diuresis. The p2mIME cells survive an acute increase in NaCl and urea to 1,340 mosmol/kgH 2 O, which exceeds the tolerance of mIMCD3 cells but is still less than occurs in vivo ( 44 ). Thus part, but not all, of the greater osmotic tolerance of inner medullary cells in vivo than in cell culture is explained by slower proliferation and DNA replication.
An additional factor is the rate at which NaCl and urea increase. The most convenient way to increase osmolality is to replace the medium in a single step, and that is the way that most experiments have been performed. However, the initial changes in osmolality in vivo occur over a period of hours. The response to changes in osmolality can depend on the rate of change. When cells are exposed to a step increase or decrease in osmolality, they immediately shrink or swell. Then, they generally undergo a regulatory volume increase or decrease, which restores cell volume toward normal ( 20 ). Interestingly, when the rate of change of osmolality is slow enough, isovolumetric regulation (IVR) occurs; i.e., no measurable change occurs in cell volume, despite large cumulative changes in osmolality. For example, when the osmolality of fluid bathing isolated S2 proximal tubules is changed at 1.5 mosmol/min, they undergo IVR, maintaining a constant volume between 167 and 361 mosmol/kgH 2 O. In contrast, a step increase in this magnitude rapidly shrinks the cells, and no regulatory volume increase ensues ( 17 ). The results are similar in C6 rat glioma cells exposed to step vs. gradual increases in osmolality ( 23 ). With these considerations in mind, we developed a system for gradually changing the osmolality bathing p2mIME cells ( 5 ). Using this system, when osmolality is increased from 640 to 1,640 mosmol/kgH 2 O by addition of NaCl and urea in a single step, only 30% of cells survive for 24 h. However, when the same increase is made linearly over 20 h, 89% of the cells remain viable 24 h later. Thus gradual changes in osmolality, as in vivo, allow cells to survive much greater changes than do the step changes routinely used in cell culture experiments.
In the present studies, we investigated why a gradual increase in NaCl and urea is better tolerated than a step increase by measuring the mRNA expression of genes whose expression is known to be affected by NaCl and urea, and which in many cases had been previously shown to protect against osmotic stress. The genes include those involved in accumulation of compatible organic osmolytes and those for heat shock proteins (HSPs), as follows.
The accumulation of compatible organic osmolytes affords protection against hypertonicity (e.g., from high NaCl) ( 9, 10 ). The predominant renal inner medullary-compatible organic osmolytes are sorbitol, glycine betaine (betaine), myo- inositol (inositol), glycerophosphocholine, and taurine. The accumulation of these organic osmolytes protects cells by normalizing cell volume and ionic strength in the face of hypertonicity. The accumulation of sorbitol depends on increased mRNA of aldose reductase (AR), an enzyme that catalyzes its synthesis from glucose, betaine on increased mRNA of its transporter BGT1, and taurine on increased mRNA of its transporter Tau. Tonicity-responsive enhancer binding protein (TonEBP) is the transcription factor responsible for hypertonicity-induced increases in the abundance of these mRNAs. Hypertonicity increases TonEBP mRNA ( 10 ).
Expression of HSPs protects renal medullary cells from high salt and urea ( 3 ). In vivo, there is a gradient of abundance of HSPs paralleling the corticomedullary osmotic gradient, and HSP abundance rises during antidiuresis associated with the increased inner medullary osmolality. Greater expression of HSPs enhances tolerance of Madin-Darby canine kidney (MDCK) cells for high urea. High urea, alone, does not increase HSP expression in MDCK cells, but high NaCl does, and high NaCl increases tolerance of MDCK and p2mIME ( 44 ) cells for high urea. High NaCl reduces the viability of HSP70.1 -deficient embryonic fibroblasts, and chronic excessive ingestion of NaCl increases apoptosis in the renal medulla of HSP70.1 -deficient mice ( 34 ).
In the present study, we compared the effect of a gradual increase vs. a step increase in NaCl and urea on mRNA abundance of these genes, finding that the gradual increase leads to greater expression of protective genes.
MATERIALS AND METHODS
Tissue culture. Mouse inner medullary epithelial cells were cultured as previously described ( 44 ). Briefly, kidneys were harvested from mice (Taconic Farms, Germantown, NY). Renal inner medullas and papillas were dissected, minced (1-2 mm), and digested in 640 mosmol/kgH 2 O (80 mM urea and 130 mM NaCl added) DMEM-F-12, containing collagenase B (Roche, Indianapolis, IN) and hyaluronidase (Worthington Biochemical, Lakewood, NJ). Cells were washed and resuspended in 640 mosmol/kgH 2 O growth medium (10% fetal bovine serum, 45% DMEM, 45% Coon's improved medium F-12, 10 mM HEPES, pH 7.5, 5 mg/l transferrin, 10 nM selenium, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo- L -thyronine, 2 mM L -glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin sulfate) and maintained at 37°C in 5%CO 2. Passage 2 cells were plated on 26-mm cell culture inserts (Becton Dickinson Labware, Bedford, MA) for the experiments.
Step and linear increases in osmolality. The procedures were previously described ( 5 ). Briefly, when the cells were confluent on cell culture inserts, the medium was changed to serum-free 640 mosmol/kgH 2 O medium (45% DMEM, 45% Coon's improved medium F-12, 10 mM HEPES, pH 7.5, 5 mg/l transferrin, 10 nM selenium, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo- L -thyronine, 2 mM L -glutamine, 5 mg/l insulin) for 48 h. Then, for a step increase to 1,040, 1,240, or 1,640 mosmol/kgH 2 O, the medium was changed both inside and outside of the insert by the addition of NaCl alone to 450 mM, urea alone to 400 or 600 mM, or NaCl to 400 mM and urea to 800 mM for 19 or 20 h. To achieve a linear increase, the cell culture inserts were mounted in chambers with a constant flow of medium bathing the lower surface of the porous supports. The osmolality of this medium was increased linearly to 1,040 or 1,640 mosmol/kgH 2 O over 19 or 20 h, using a gradient mixer. The osmolality within the insert increases with a similar time course ( 5 ). After the linear increase in NaCl alone to 1,040 mosmol/kgH 2 O, cells were also maintained at 1,040 mosmol/kgH 2 O for an additional 19 h.
Preparation of total RNA and cDNA. Total RNA was isolated from p2mIME cells with RNeasy spin columns (Qiagen, Valencia, CA) as recommended by the manufacturer. To eliminate DNA contamination, the RNA was treated for 15 min with RNase-free DNase, using RNeasy columns (Qiagen). Two micrograms of total RNA were reverse transcribed with random hexamers, using a Taq Man Kit (Applied Biosystems, Foster City, CA).
Real-time PCR. cDNA was quantitated with the ABI Prism 7900 Sequence Detection System (Applied Biosystems) following the manufacturer's recommendations. This system utilizes the 5' nuclease activity of Taq DNA polymerase to generate the signal. Briefly, gene-specific primers and oligonucleotide probes containing a 5' fluorescent dye, 6-FAM, and a 3' quencher, TAMRA ( Taq Man probes), were designed using Primer Express software (Applied Biosystems). The primers all span introns, except for intronless genes, namely, HSP70.1 and HSP70.3. Primer pairs and probe sequences from 5' to 3' are listed for forward primers (F), reverse primers (R), and Taq Man probes (P) in Table 1. Multiplex PCR was performed using Taq Man PCR Master Mix (Applied Biosystems) to which was added both the specific primers and probes and 18S rRNA primers and 18S probe, labeled with the fluorescent dye VIC. The coamplified 18S cDNA serves as an internal control for reverse transcription and cDNA loading. Triplicates of each sample were analyzed in each PCR run. The PCR reaction sequence was 2 min at 50°C for optimal AmpErase UNG enzyme activity; 10 min at 95°C to activate Ampli- Taq Gold DNA polymerase; then 40 cycles of 95°C for 15 s and 60°C for 1 min. The results were analyzed using the ABI Prism 7900 system software. The AR, BGT1, Tau, and TonEBP PCR products were verified by sequencing (Amplicon Express, Pullman, WA). Representative amplification plots are shown in Fig. 1 D.
Table 1. Primers and probes used for real-time PCR
Fig. 1. Effect of rate of increase in NaCl and/or urea on mRNA abundance of tonicity-responsive enhancer binding protein ( TonEBP ), aldose reducatse ( AR ), betaine transporter ( BGT1 ), and taurine transporter ( Tau ). Osmolality was initially at 640 mosmol/kgH 2 O. A : NaCl was added from 640 to 1,040 mosmol/kgH 2 O. mRNA was measured 19 h after NaCl was added in a single step, after 19 h of a linear increase in NaCl, or while the osmolality was still elevated 19 h after completion of the linear increase. B : addition of equiosmolal concentrations of NaCl and urea from 640 to 1,640 mosmol/kgH 2 O or removal of some of the NaCl and all of the urea to lower osmolality from 640 to 300 mosmol/kgH 2 O. mRNA was measured 20 h after the step increase, after 20 h of a linear increase, or 48 h after the step decrease. C : osmolality was increased by adding urea in a single step, and mRNA was measured 20 h later. Values are means ± SE; n = 3 or 4. * P < 0.05 compared with 640 mosmol/kgH 2 O. D : representative real-time amplification plot of AR mRNA. Dashed line, threshold.
Analysis of real-time PCR data. The ABI Prism 7900 system records the number of PCR cycles (Ct) required to produce an amount of product equal to a constant threshold value, set to be reached during the exponential phase of the PCR reaction. As previously described ( 8 ), relative mRNA abundance was calculated from the real-time PCR data using the following principles. 1 ) By definition the number of specific cDNA molecules at the threshold ( N Ct ) is constant for a given cDNA, independent of the number of cycles that it takes to reach it. 2 ) For a specific cDNA the ratio N (exp) i / N (cont) i is independent of i, assuming only that the efficiency ( E ) of PCR for a specific template is constant, where i is the cycle number, and N ( X ) i is the number of specific cDNA molecules in a sample ( X = control or experimental) at cycle i. 3 ) The ratio of the number of specific cDNA molecules at a cycle, Ct, to the number at another cycle, i, is Ni / N Ct = 1/ E (Ct- i ).
To compare control and experimental results, we normalized both to the number of specific molecules at an arbitrary cycle, I, chosen for convenience to be the largest whole number that is less than any of the experimental values of Ct. Then, we calculated N ( X ) I / N Ct for each sample. Experimental results are presented as the percentage of the corresponding control value.
Measurement of total RNA per cell. p2mIME cells (3 x 10 5 ) were seeded in 640 mosmol/kgH 2 O growth medium on 26-mm cell culture inserts and allowed to grow for 3 days, followed by 48 h in serum-free medium (see above). Then, osmolality was kept at 640 or was increased to 1,640 mosmol/kgH 2 O in a single step (see above) for 20 h. Cells on some inserts were counted with a Neubauer hemocytometer after trypsinization. Total RNA was measured after extraction from other cells grown on other inserts the same way, and total RNA per cell (pg/cell) was calculated.
Western blot analysis. Cultures were washed twice with ice-cold phosphate-buffered saline, adjusted with NaCl and urea to the same osmolality as the medium. Then they were lysed with mammalian protein extraction reagent (Pierce, Rockford, IL) to which was added protease inhibitor (Roche). Extracts were centrifuged at 15,000 rpm for 10 min. Protein concentration was measured by using the BCA protein assay (Pierce). An equal amount of protein (0.5 µg) was loaded onto each lane, and electrophoresis was performed using 12% acrylamide-Tris-glycine gels. Immunodetection utilized polyclonal primary antibodies against aldose reductase (kindly provided by Dr. Peter Kador) or HSP70 (Calbiochem, San Diego, CA), and mouse anti-goat (Pierce) or mouse anti-rabbit IgG (cell signaling) secondary antibodies, conjugated with horseradish peroxidase.
Statistical analysis. Statistical analysis was performed using GraphPad Instat after log transformation of the data. Results are expressed as mean ± SE ( n = no. of independent experiments). Differences were considered significant for P < 0.05, determined with the paired t -test or with one-way ANOVA followed by Dunnett's multiple comparison test for separation of significant means.
RESULTS
Effect of rate of change of osmolality on mRNA abundance of TonEBP, AR, BGT1, and Tau. These genes are involved in tonicity-dependent accumulation of compatible organic osmolytes ( 9, 10 ). Hypertonicity increases BGT1, AR, and/or Tau mRNAs in cultures of immortalized cells and in vivo. Transcription of AR and BGT1 increases, mediated by hypertonicity-induced activation of TonEBP. Hypertonicity increases TonEBP mRNA in MDCK cells, reaching a threefold maximum at 12 h, followed by a decrease ( 41 ). To test the effect of rate of change of tonicity on abundance of these mRNAs, we increased NaCl either in a single step or linearly over 19 h, raising osmolality of the medium bathing p2mIME cells from 640 to 1,040 mosmol/kgH 2 O ( Fig. 1 A ). We measured mRNA 1 ) 19 h after the step increase, 2 ) after 19 h of a linear increase when the osmolality had just reached 1,040 mosmol/kgH 2 O, and 3 ) 19 h after the linear increase had reached 1,040 mosmol/kgH 2 O ( Fig. 1 A ). AR and BGT1 mRNA levels increase approximately twice as much with the linear increase as with the step increase. The higher level with linear increase is sustained for at least an additional 19 h at 1,040 mosmol/kgH 2 O. At that time AR mRNA remains increased to 1,317 ± 107% of its initial value at 640 mosmol/kgH 2 O and BGT1 to 1,319 ± 248%. After 19 h of high NaCl, TonEBP mRNA is unchanged ( Fig. 1 A ), consistent with the previous observation that it increases to a peak after 6 h of high salt, then falls ( 11 ).
NaCl and urea in combination increase in the renal inner medulla during antidiuresis in vivo ( 1 ). To investigate the effect of rate of increase in NaCl and urea in combination on expression of AR, BGT1, Tau, and TonEBP, we increased the osmolality of the medium bathing confluent p2mIME cells either acutely, in a single step, or gradually over 20 h from 640 to 1,640 mosmol/kgH 2 O by adding NaCl and urea in combination ( Fig. 1, B and D ). Although TonEBP and AR mRNAs rise after 20 h of linear increase in osmolality, they fall substantially after a step increase. BGT1 mRNA does not change after the linear increase in osmolality and decreases substantially after the step increase. Tau decreases substantially after both the linear and step increase in osmolality.
For comparison with the many previous experiments in which the baseline osmolality was 300 mosmol/kgH 2 O, we decreased osmolality in a single step from 640 to 300 mosmol/kgH 2 O by substituting a medium containing no excess urea or NaCl, and we measured mRNA levels 48 h later ( Fig. 1 B ). Under those conditions, AR and BGT1 mRNA levels decrease substantially, but those of TonEBP and Tau do not change.
Thus levels of AR and BGT1 mRNAs rise after a step increase in NaCl ( Fig. 1 A ) but fall after a step increase in NaCl plus urea ( Fig. 1 B ). Urea could account for the difference. In agreement with this possibility, high urea previously was observed to block tonicity-dependent increases in TonEBP and AR mRNA ( 38 ). To confirm this, we tested the effect of a step increase in urea ( Fig. 1 C ). Increasing osmolality from 640 to 1,040 or 1,240 mosmol/kgH 2 O in a single step substantially decreases the level, not only of AR and TonEBP mRNA, but also of BGT1 and Tau mRNA.
Effect of rate of change of osmolality on expression of HSP70 and OSP94 mRNAs. Hypertonicity increases protein and mRNA abundance of HSP70, both in the absence ( 6, 33 ) and presence ( 32 ) of added urea. Increased expression of HSP70 protein protects the cells from harmful effects of high urea ( 25, 26 ) and makes them more adaptable to changes in osmolality ( 32 ). Analysis of these effects is complicated by the existence of two HSP70 genes, HSP70.1 and HSP70.3, whose protein products are identical but whose regulatory regions differ. For simplicity and consistency, we will refer to these genes according to their designation in mice ( Table 1 ), which is the species we studied, and will not use the different (and sometimes conflicting) names given to their homologues in other species ( 40 ). Heat shock increases expression of both HSP70.1 and HSP70.3. The HSP70.1 gene contains 5'-flanking TonEs, but the HSP70.3 gene does not ( 42 ). Accordingly, hypertonicity was reported to increase the abundance of HSP70.1 mRNA, but not of HSP70.3 mRNA ( 34, 42 ). OSP94 is a member of the HSP110 subfamily of HSP70 genes ( 12, 15 ). Its mRNA expression is high in mouse inner medulla, and is induced in mIMCD3 cells by hypertonicity and heat shock, but not by high urea ( 31 ). In the present studies, we measured HSP70.1, HSP70.3, and OSP94 mRNAs in p2mIME cells following step or linear increases of NaCl and/or urea.
Nineteen hours after osmolality was increased in a single step from 640 to 1,040 mosmol/kgH 2 O by adding NaCl, HSP70.1, HSP70.3, and OSP94 mRNA levels are increased several-fold in p2mIME cells ( Fig. 2 A ). The result is essentially the same when osmolality is increased linearly over 20 h from 640 to 1,640 mosmol/kgH 2 O by adding NaCl and urea ( Fig. 2 B ). However, 20 h after a step increase to the same osmolality, the increases in HSP70.1 and HSP70.3 mRNA levels are much smaller than when NaCl is added alone, and OSP94 mRNA decreases substantially. To evaluate whether it is urea that accounts for the smaller increases in HSP70.1 and HSP70.3 mRNA with step vs. linear increase in osmolality and the decrease in OSP94 mRNA, we tested the effect of step increase in urea alone. Adding urea in a single step to increase osmolality from 640 to 1,040 mosmol/kgH 2 O decreases mRNA abundance of all three heat shock genes ( Fig. 2 C ). Adding urea in a single step to increase osmolality from 640 to 1,240 mosmol/kgH 2 O decreases mRNA abundance of HSP70.1 and OSP94 but increases HSP70.3 mRNA. Thus urea can account for much of the lesser elevation of HSP mRNAs with step vs. linear increase in NaCl plus urea.
Fig. 2. Effect of rate of increase in NaCl and/or urea on mRNA abundance of heat shock proteins ( HSP70.1 and HSP70.3 ) and osmotic stress protein 94 ( OSP94 ). Osmolality was initially at 640 mosmol/kgH 2 O. A : NaCl was added in a single step from 640 to 1,040 mosmol/kgH 2 O, and mRNA was measured 19 h later. B : addition of equiosmolal concentrations of NaCl and urea from 640 to 1,640 mosmol/kgH 2 O or removal of some of the NaCl and all of the urea to lower osmolality from 640 to 300 mosmol/kgH 2 O. mRNA was measured 20 h after the step increase, after 20 h of a linear increase, or 48 h after the step decrease. C : osmolality was increased by adding urea in a single step, and mRNA was measured 20 h later. Values are means ± SE; n = 3 or 4. * P < 0.05 compared with 640 mosmol/kgH 2 O.
When osmolality is decreased from 640 to 300 mosmol/kgH 2 O for 48 h, HSP70.1 mRNA is greatly reduced and HSP70.3 and OSP94 mRNAs are reduced to a lesser extent ( Fig. 2 B ). This reduction is reminiscent of the corticomedullary gradient of HSP expression in vivo, which parallels the renal osmolality gradient lowest in the renal cortex, highest in the renal papilla ( 3 ).
Effect of rate of change of osmolality on expression of Egr-1, GADD153, and heme oxygenase-1 mRNAs. Egr-1 is an immediate-early response gene. GADD153 is a DNA damage- and growth arrest-inducible gene, encoding the oxidative stress-responsive member of the CCAAT/enhancer-binding protein transcription factor family. Heme oxygenase-1 ( HO-1 ) is induced in response to prooxidant stimuli as well as other stressors. These three genes have in common that the abundance of their mRNA and protein is increased by high urea, although in mIMCD3 cells, HO-1 and GADD153 mRNA and protein increase only over a limited concentration range of high urea ( 37 ).
We first tested the effects on these genes of adding NaCl and urea together. In p2mIME cells, increasing osmolality in a single step from 640 to 1,640 mosmol/kgH 2 O by adding urea and NaCl greatly increases Egr-1 mRNA ( Fig. 3 A ). The slower, linear increase in osmolality increases Egr-1 mRNA to a lesser extent ( Fig. 3 A ). A step decrease in osmolality from 640 to 300 mosmol/kgH 2 O, which is mainly a decrease in NaCl concentration, does not affect Egr-1 mRNA abundance ( Fig. 3 A ). The effects on GADD153 mRNA abundance of changing urea and NaCl are similar to those on Egr-1, except that GADD153 mRNA increases with a fall, as well as with an increase in osmolality. In contrast, although a linear increase in NaCl and urea elevates HO-1 mRNA, a step increase reduces it, and it is unaffected by a decrease in osmolality ( Fig. 3 A ).
Fig. 3. Effect of rate of increase in NaCl and/or urea on mRNA abundance of Egr-1, GADD153, heme oxygenase-1 ( HO-1 ), and Herp. Osmolality was initially at 640 mosmol/kgH 2 O. A : addition of equiosmolal concentrations of NaCl and urea from 640 to 1,640 mosmol/kgH 2 O or removal of some of the NaCl and all of the urea to lower osmolality from 640 to 300 mosmol/kgH 2 O. mRNA was measured 20 h after the step increase, after 20 h of a linear increase, or 48 h after the step decrease. B : osmolality was increased by adding urea in a single step, and mRNA was measured 20 h later. Values are means ± SE; n = 4. * P < 0.05 compared with 640 mosmol/kgH 2 O.
As in previous studies with mIMCD3 cells, a step increase in urea concentration from 640 to 1,040 mosmol/kgH 2 O elevates Egr-1, GADD153, and HO-1 mRNA abundances in p2mIME cells ( Fig. 3 B ). The result is similar at 1,240 mosmol/kgH 2 O, except that HO-1 mRNA decreases.
Herp mRNA. An accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response that increases molecular chaperones and enzymes that assist in protein folding in the ER, thus affecting cellular survival and apoptosis. The unfolded protein response (UPR) increases transcription of a characteristic set of target genes, including Herp ( 13 ). Urea denatures proteins and nucleic acids. To determine whether high urea or urea plus NaCl produces ER stress and the UPR, we measured Herp mRNA. Increasing osmolality by adding NaCl and urea ( Fig. 3 A ) or urea alone ( Fig. 3 B ) substantially decreases Herp mRNA rather than increasing it. Thus we find no evidence that high urea induces the UPR, as previously inferred from the finding that urea has no effect on the expression of the UPR-inducible protein GRP78 ( 46 ). Reducing osmolality from 640 to 300 mosmol/kgH 2 O does not significantly affect Herp mRNA ( Fig. 3 A ).
Effect of high NaCl and/or urea on expression of -actin, myosin 1B, and hypoxanthine-guanine phosphoribosylphosphotransferase mRNA. Our measurements were intended to compare mRNA abundance between experimental conditions. To achieve this, we reverse transcribed equal amounts of total RNA and measured specific cDNAs by real-time PCR. Such comparisons are commonly validated by measuring in the same RNA samples the abundance of gene products that are not expected to change, the so-called "housekeeping genes" ( 39 ), which requires, of course, knowledge that expression of the housekeeping genes themselves does not change, making the process somewhat circular. Because expression of housekeeping genes may vary, caution has been urged in their use to normalize measurements of mRNA abundance ( 4, 39 ). Housekeeping genes used for this process include -actin, myosin 1B, and hypoxanthine-guanine phosphoribosylphosphotransferase ( HPRT ). We found that high NaCl and/or urea significantly changes the abundance of the cDNAs for these genes ( Fig. 4 ), which obviates their use for normalization. Step or linear increase in NaCl and/or urea above 640 mosmol/kgH 2 O significantly decreases all three ( Fig. 4, A -C ), while a step decrease from 640 to 300 to mosmol/kgH 2 O increases -actin and myosin 1B, but not HPRT. Therefore, we used a different strategy to validate the results.
Fig. 4. Effect of rate of increase in NaCl and/or urea on abundance of -actin, myosin 1B, and hypoxanthine-guanine phosphoribosylphosphotransferase ( HPRT ) mRNA and on 18S rRNA abundance. Osmolality was initially at 640 mosmol/kgH 2 O. A : addition of equiosmolal concentrations of NaCl and urea from 640 to 1,640 mosmol/kgH 2 O or removal of some of the NaCl and all of the urea to lower osmolality from 640 to 300 mosmol/kgH 2 O. mRNA or rRNA was measured 20 h after the step increase, after 20 h of a linear increase, or 48 h after the step decrease. B : osmolality was increased by adding urea in a single step, and mRNA or rRNA was measured 20 h later. C : NaCl was added in a single step from 640 to 1,040 mosmol/kgH 2 O, and mRNA or rRNA was measured 19 h later. Values are means ± SE; n = 3 or 4. * P < 0.05 compared with 640 mosmol/kgH 2 O.
Validation of RT-PCR measurement of specific mRNAs. To validate the method, we compared the amounts of total RNA per cell and of 18S RNA in different osmotic conditions. Twenty hours after a step increase in osmolality to 1,640 mosmol/kgH 2 O, total RNA per cell is 33.3 ± 9.4 pg compared with 43.7 ± 13.4 pg for the control, which is not significantly different ( P < 0.05). Of total RNA, 80% is rRNA. Therefore, a lack of significant change in total RNA per cell implies no important change in rRNA. A major component of rRNA, 18S rRNA, was measured in each real-time PCR reaction. It does not change in any experimental condition ( Fig. 4 ), confirming that reverse transcription and sample loading were equivalent between control and experimental conditions. This provides confidence in the measured changes in specific mRNAs, including those for the housekeeping genes.
Effect of rate of change of osmolality on expression of AR and HSP70 protein. Changes in expression of mRNAs are not necessarily accompanied by proportional changes in the abundance of the proteins that are translated from them. Therefore, we also measured by semiquantitative Western blot analysis the abundance of two proteins ( Fig. 5 ) known to be protective against high NaCl or urea. Sorbitol, accumulated as a result of increased abundance of AR protein, enhances survival of renal cells exposed to high NaCl ( 43 ), and HSP70 protein enhances survival of renal cells exposed to high NaCl and urea ( 3, 26 ). Because HSP70.1 and HSP70.3 proteins are identical, the antibody that was used recognizes both. Both AR ( Fig. 1 B ) and HSP70 ( Fig. 2 B ) mRNAs increase more with a linear than with a step increase when osmolality is increased from 640 to 1,640 mosmol/kgH 2 O by adding NaCl and urea. The same is true of the proteins ( Fig. 5 ). In particular, HSP70 protein increases 2.6-fold with a linear increase in osmolality, but does not increase significantly with a step increase.
Fig. 5. Effect of rate of increase in NaCl and urea on protein abundance of AR and HSP70. Osmolality was increased by addition of equiosmolal concentrations of NaCl and urea from 640 to 1,640 mosmol/kgH 2 O. Protein was extracted 20 h after the step or linear increase. A and C : representative Western blots. B and D : densitomery. Values are means ± SE; n = 3. * P < 0.05 compared with 640 mosmol/kgH 2 O.
DISCUSSION
We previously found that p2mIME cells survive higher levels of urea and NaCl when the solutes increase linearly, over several hours, than when they increase in a single step ( 5 ). In the present study, we searched for a possible reason for this difference, by comparing the effect of a gradual increase vs. a step increase in NaCl and/or urea on mRNA abundance of several classes of genes, namely, 1 ) those involved in osmolality-induced accumulation of protective organic osmolytes, 2 ) HSP genes, 3 ) urea-induced genes, and 4 ) housekeeping genes.
mRNAs of genes involved in osmolality-induced accumulation of protective organic osmolytes include TonEBP, AR, Tau, and BGT1 (see the beginning of this study). A linear increase in NaCl elevates AR and BGT1 mRNAs much more than does a step increase ( Fig. 1 A ). Furthermore, a step increase in urea and NaCl reduces TonEBP, AR, Tau, and BGT1 mRNAs ( Fig. 1 B ). Because urea alone decreases these mRNAs ( Fig. 1 C ), it apparently is responsible for reducing their abundance when NaCl and urea in combination are added in a single step ( Fig. 1 B ). We previously observed that urea reduces the content of betaine in MDCK cells ( 24 ) and of both betaine and sorbitol in PAP-HT25 cells ( 21 ). Furthermore, urea reduces hypertonicity-induced increase in AR and TonEBP mRNA in mIMCD3 cells ( 38 ). Evidently, a step increase in urea reduces RNA expression of the osmoprotective genes, TonEBP, AR, and BGT1, regardless of whether NaCl also increases. In contrast, a gradual, linear increase in NaCl and urea in combination does not reduce RNA expression of these genes, and, in fact TonEBP and AR mRNAs increase, associated with the greater survival of cells that follows a linear increase ( 5 ). We conclude that a gradual, linear increase in NaCl and urea ameliorates suppression by a step increase in urea of the tonicity-induced activation of genes responsible for protective accumulation of compatible organic osmolytes.
The marked activation of these genes by a slow increase in tonicity has implications for the signal that is involved. When cells are exposed to hypertonicity in a single step, they shrink immediately. Thus decreased cell volume could be a signal for induction of organic osmolyte accumulation under those conditions. However, when osmolality increases gradually (1.5-3.0 mosmol·kg -1 ·min -1 ), IVR occurs and there is no measurable change in cell volume ( 17, 23 ). IVR involves electrolyte uptake by the combined action of the Na-K-2Cl cotransporter and Na-K-ATPase, which increases intracellular ionic strength ( 23 ). It was not practical for us to measure cell volume in our experiments. However, when we added NaCl alone, the rate of increase in osmolality was only 0.35 mosmol·kg -1 ·min -1, a rate much less than that which did not result in any change in cell volume in two types of cells previously studied. Nevertheless, the abundance of AR and BGT1 mRNA increased 6- to 18-fold ( Fig. 1 A ). Thus it seems unlikely that any decrease in cell volume signaled the increase in AR and BGT1 mRNAs that we observed after the slow, linear increase in NaCl. Then, what is the signal? Increased abundance and activity of AR account for tonicity-induced accumulation of sorbitol ( 9 ). We previously found that in PAP-HT25 cells the induction of AR activity in response to hypertonicity is closely related to the sum of intracellular sodium and potassium concentrations and not to cell volume ( 9 ). The interpretation was that an intracellular concentration of sodium plus potassium salts (or ionic strength) is the signal by which hypertonicity induces accumulation of organic osmolytes. In support of this hypothesis, hypertonicity-induced TonEBP activity also correlates with intracellular ionic strength in MDCK cells ( 28 ). However, increased intracellular ionic strength is not sufficient to increase TonEBP activity. Intracellular ionic strength is increased by high extracellular potassium concentration and by ouabain in MDCK cells without activating TonEBP ( 28 ). Either some factor in addition to intracellular ionic strength is involved or high potassium and ouabain have additional effects, such as the cell volume increase that they cause, that subvert the signal.
mRNAs of genes for HSPs. HSPs ( 2 ) are a group of highly conserved proteins, which are expressed constitutively and/or induced by stress. Constitutively expressed HSPs participate in protein folding and assembly, elimination of misfolded proteins, and stabilization of newly synthesized protein in various intracellular compartments. Expression of inducible HSPs is evoked by various stresses, many of which denature proteins ( 19 ). The HSPs enhance cellular survival and aid cellular recovery by acting as molecular chaperones that limit or correct damage to proteins. HSPs are abundant in renal medullas, and high NaCl increases expression of HSPs, protecting cells from damaging effects of high urea (see the beginning of this study). There are two HSP70 genes that have identical open reading frames but that differ in their regulation. The HSP70.1 gene contains ORE/TonEs in its 5'-flanking region, and its expression is increased both by hypertonicity and heat shock in mIMCD3 cells ( 42 ). HSP70.3 does not contain ORE/TonEs, and its expression is increased by heat but not by hypertonicity in mIMCD3 cells ( 42 ). However, we now find that a step increase in NaCl elevates both HSP70.1 and HSP70.3 mRNAs in p2mIME cells ( Fig. 2 A ). Apparently, a step increase in NaCl can increase HSP70.3 mRNA by mechanisms independent of an effect of TonEBP on known 5'-flanking ORE/TonEs. We previously proposed one such mechanism, which is that hypertonicity might damage proteins, providing a signal to increase HSP70 ( 33 ). Another possible mechanism involves double-stranded RNA-dependent protein kinase (pkr)-dependent stabilization of HSP70 mRNA. Pkr-mediated stabilization of HSP70 mRNA is adenosine-uridine rich element (ARE) dependent and can be enhanced by heat shock, resulting in accumulation of HSP70 mRNA ( 46 ). ARE sequences are located in the 3'-untranslated region (UTR) of some mRNAs. The existence of AREs destabilize ARE-containing mRNAs. HSP70.1 mRNA contains two consensus ARE (AUUUA) sequences in its 3'-UTR region, but we find none in HSP70.3. Thus this mechanism could be involved in a hypertonicity-induced increase in HSP70.1 mRNA, but an involvement is less likely for HSP70.3. Adding 300 mM urea, which MDCK cells survive, does not increase expression of HSPs ( 27 ). Our present results are similar. Adding 400 mM urea (total osmolality 1,040 mosmol/kgH 2 O; Fig. 2 C ) to p2mIME cells in a single step, which the cells survive ( 44 ), reduces HSP70.1, HSP70.3, and OSP94 mRNAs. In contrast, although adding 600 mM urea (total osmolality 1,240 mosmol/kgH 2 O; Fig. 2 C ) to p2mIME cells in a single step kills 20% of the cells ( 44 ) and reduces both HSP70.1 and OSP94 mRNAs, it increases HSP70.3 mRNA. HSP70 is induced by denaturation of cellular proteins ( 19 ), and urea is a potent denaturant ( 16 ), which may explain instances of induction of HSPs by high levels of urea.
In the present study, we found that larger increases in HSP mRNAs occur after a gradual, linear increase in NaCl and urea than after a step increase ( Fig. 2 B ). After the gradual, linear increase, HSP70.1, HSP70.3, and OSP94 mRNA increase fiveto ninefold. In contrast, after the step increase, OSP94 mRNA decreases, HSP70.1 is little changed, and HSP70.3 increases only threefold. It is the urea that apparently accounts for the difference. A step increase in NaCl alone increases HSP70.1, HSP70.3, and OSP94 mRNAs ( Fig. 2 A ). In contrast, a step increase in urea from 640 to 1,040 mosmol/kgH 2 O reduces HSP70.1, HSP70.3, and OSP94 mRNAs, and a step increase in urea from 640 to 1,240 mosmol/kgH 2 O reduces HSP70.1 and OSP94 mRNAs ( Fig. 2 C ). Given that expression of HSPs protects cells against osmotic stress, the greater rise in HSP mRNAs ( Fig. 2 ) and HSP70 protein ( Fig. 5 ) after a gradual, linear increase in NaCl and urea than after a step increase could contribute to the greater survival after the linear increase.
This apparently detrimental effect of a step increase in urea in combination with NaCl is surprising in view of previous studies showing that a step increase in a combination of NaCl and urea is less damaging to renal cells than a step increase in either alone ( 27, 30, 45 ). Protection against urea by NaCl was explained by the effect of HSP70, which was found to be increased by high NaCl but not high urea ( 27 ). The present studies differ from the previous ones in that we used p2mIME cells, which endure acute effects of the high concentrations of combined salt and urea that exist in the renal inner medulla but which are lethal when acutely applied to the immortalized cells previously studied. Thus a step addition of 50-200 mM urea protects mIMCD3 cells from apoptosis induced by adding 200-400 mosmol/kgH 2 O of NaCl, 200 mM urea is proapoptotic ( 45 ), inhibiting tonicity-induced TonEBP activity and expression of AR ( 38 ). The step increases that we studied were 400 mM or more of urea, well above both the level found to be protective in mIMCD3 cells and the higher level found to be detrimental. It is telling that the detrimental effects of urea that occur with step increases are not apparent during a gradual, linear increase in combined NaCl and urea. Evidently, the cells accommodate better to the more natural, gradual increase for reasons that probably include the enhanced expression of protective genes.
Egr-1, GADD153, and HO-1 mRNAs and proteins are increased by high NaCl or urea in mIMCD3 cells ( 37 ). Egr-1 apparently participates in signaling for both cellular proliferation and apoptosis ( 35 ). GADD153 (also called CHOP) is implicated in programmed cell death ( 47 ). A linear increase in NaCl and urea in combination causes less increase in Egr-1 and GADD153 mRNA than does a step increase ( Fig. 3 A ).
HO-1 is induced by diverse stresses. It protects cells by its antioxidant, antiapoptotic, and anti-inflammatory actions ( 22 ). In the kidney, there is a gradient of HO-1 mRNA and protein, highest in the renal inner medulla and lowest in the renal cortex ( 48 ). HO-1 is induced by either high NaCl or urea in mIMCD3 cells ( 36 ). Inhibition of HO-1 by the HO-1 inhibitor ZnDPBG reduces tolerance of mIMCD3 cells for high NaCl but does not affect tolerance for high urea. We now find that, in agreement with the results using mIMCD3 cells, a step increase in urea raises HO-1 mRNA in p2mIME cells ( Fig. 3 B ). However, when NaCl and urea in combination are increased in p2mIME cells, the results differ, depending on whether the increase is a single step or a gradual, linear increase. A step increase in both NaCl and urea reduces HO-1 mRNA in p2mIME cells ( Fig. 3 A ), but a linear increase in urea and NaCl in combination raises HO-1 mRNA ( Fig. 3 A ). This difference in HO-1 expression could contribute to the greater tolerance of p2mIME cells for a gradual, linear increase in NaCl and urea in combination.
ACKNOWLEDGMENTS
We are very grateful to Dr. Peter F. Kador for the aldose reductase antibody.
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作者单位：Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung,and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603