Long-term adaptation of renal cells to hypertonicity: role of MAP kinases and Na-K-ATPase

J. M. Capasso, C. J. Rivard, and T. Berl

Division of Nephrology, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Renal cells in culture have low viability when exposed to hypertonicity. We developed cell lines of inner medullary collecting duct cells adapted to live at 600 and 900 mosmol/kgH2O. We studied the three modules of the mitogen-activated protein (MAP) kinase family in the adapted cells. These cells had no increase in either extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, or p38 MAP kinase protein or basal activity. When acutely challenged with further increments in tonicity, they had blunted activation of these kinases, which was not due to enhanced phosphatase activity. In contrast, the cells adapted to the hypertonicity displayed a marked increment in Na-K-ATPase expression (5-fold) and ouabain-sensitive Na-K-ATPase activity (10-fold). The changes were reversible on return to isotonic conditions. Replacement of 300 mosmol/kgH2O of NaCl by urea in cells adapted to 600 mosmol/kgH2O resulted in marked decrement in Na-K-ATPase and failure to maintain the cell line. Replacement of NaCl for urea in cells adapted to 900 mosmol/kgH2O did not alter either Na-K-ATPase expression, or the viability of the cells. The in vivo modulation of Na-K-ATPase was studied in the renal papilla of water-deprived mice (urinary osmolality 2,900 mosmol/kgH2O), compared with that of mice drinking dextrose in water (550 mosmol/kgH2O). Increased water intake was associated with a ~30% decrement in Na-K-ATPase expression (P < 0.02, n = 6), suggesting that this enzyme is osmoregulated in vivo. We conclude that whereas MAP kinases play a role in the response to acute changes in tonicity, they are not central to the chronic adaptive response. Rather, in this setting there is upregulation of other osmoprotective proteins, among which Na-K-ATPase appears to be an important component of the adaptive process.

mitogen-activated protein; kidney cells; hyperosmolality; sodium pump


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE ABILITY OF RENAL CELLS to survive in the hypertonic environment of the inner medulla is dependent on the early activation of ion transport systems that allow for regulatory volume increases (RVI) and the subsequent accumulation of organic osmolytes (4). The tonicity to which cells in the renal inner medulla are exposed can change over a broad range in the face of varying water intake. Even under conditions of water diuresis, the inner medulla is never less than isotonic, and because random urine almost always has an osmolality greater than plasma, the cells in this area of the mammalian nephron are persistently exposed to a hypertonic environment. In an attempt to mimic the in vivo setting, cells living chronically in hypertonic conditions should be subjected to alterations in tonicity. In this regard, most studies with cultured cells have examined the response of renal cells to acute changes in surrounding tonicity starting in an isotonic environment rather than their properties in a persistently hypertonic setting. Studies in a hypertonic setting have been further limited by the very low ex vivo survival of these cells when subjected to osmolalities >500 mosmol/kgH2O (5, 21, 27).

Considerable attention has been directed to the delineation of signaling events that accompany the acute exposure of cells to high osmolalities. Several investigators have described the activation of all three members of the mitogen-activated protein (MAP) kinase family in response to osmotic stress (1, 9, 31). The survival of renal cells in a hypertonic environment is clearly independent of the activation of the extracellular signal-regulated kinase (ERK) pathway (1) but is adversely affected by inhibition of c-Jun NH2-terminal kinase (JNK) activation despite intact myo-inositol uptake (27). The role of p38 MAP kinase remains more controversial as its pharmacological inhibition has been reported to inhibit aldose reductase message (22) and regulate the osmotic response element (ORE) (16), but others have found intact osmoregulation in the face of p38 MAP kinase inhibition (10). Whether activation of any of these pathways represents a transient event or an integral persistent component of the cellular response to living in hypertonic media has not heretofore been explored. Similarly, the effect of further increments in tonicity on the background of a hypertonic environment on these pathways has not been previously studied.

Another cellular response that appears to be rapidly affected by changes in tonicity is the regulation of the Na-K-ATPase gene, as evidenced by accumulation of the message for this protein in Madin-Darby canine kidney (MDCK) cells (2), proximal tubule cells (30), and inner medullary collecting duct (IMCD) cells (18). Whereas the upregulation of message appears to persist for at least 24 h (18, 30), none of these studies in renal cells examined whether these effects persist beyond this time period in cells adapted to hypertonic conditions, nor whether this effect on message is reflected in the expression of the protein. Furthermore, the effects on Na-K-ATPase have not been examined in various hydration states in vivo. Our experiments were therefore undertaken to 1) develop cell lines of IMCD cells that survive and can be maintained in an ongoing cell culture system at high tonicities and 2) examine the MAP kinase pathways and Na-K-ATPase in such cells. We hypothesized that because the MAP kinase pathways are primarily responsive to acute cellular stress, their activation, unlike that of organic osmolytes, is likely to be transient and not vital to the chronic adaptive response. We also surmised that the rapid increment in Na-K-ATPase is critical to the cells when they are acutely exposed to a high-salt environment, but we considered it likely that the activation may be more persistent because the organic osmolyte transport is Na coupled and would require subsequent exit of the cation from the cell.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture and serum were from Life Technologies. Recombinant GST-c-Jun(1-79), were expressed in Escherichia coli and purified by using glutathione-agarose (Sigma) as described previously (27). All the antisera used were purchased from Santa Cruz Biotechnology, StressGen Biotechnologies, Upstate Biotechnology, and Cell Signaling Technology. Myo-[2-3H]inositol (18.3 Ci/mol) and [alpha -32P]dCTP (3,000 Ci/mmol) were from Amersham, and [gamma -32P]ATP (3,000 Ci/mmol) and [methoxy-3H]inulin (0.5 Ci/mmol) were from NEN Life Science. All other chemicals were purchased from Sigma or Fisher and were of the maximum quality available. The osmolarity of all solutions was checked with an Advanced Instruments microosmometer.

Cell culture. The established murine inner medullary collecting duct cell line (mIMCD3) cell line was provided by Dr. Steve Gullans (Boston, MA). The cells were routinely propagated in a 1:1 mixture of DMEM and Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The M1 immortalized mouse cortical collecting duct cell line was developed and kindly provided by Dr. Geza Fejes-Toth (Dartmouth Medical School, Hanover, NH). The cells were expanded and routinely grown in RPMI 1640 medium supplemented with 10% FCS, and antibiotics. Cell survival was measured by using a CellTiter cell proliferation assay (Promega Life Science) as recommended by the manufacturer.

Development of cell lines adapted to hypertonicity. IMCD3 cells were adapted to hyperosmotic conditions by stepwise increments in the concentration of the corresponding osmolytes. Briefly, at the time when the cultures were 60-70% confluent, the appropriate volume of sterile NaCl (5 M) or urea (9 M) was added to increase the osmotic pressure of the medium by 50 mosmol/kgH2O. Cells were kept in this condition until confluence was achieved, at which time they were subcultured. After survival at this higher tonicity for at least four passages, some of the cultures were frozen in liquid nitrogen, and in others the process was restarted with a further increment of 50 mosmol/kgH2O.

Measurement of MAP kinase activity. JNK activity was measured as described (1) with the following modification. After PAGE proteins were detected by copper staining (13), the bands corresponding to c-Jun were excised, incubated in scintillation vials with 5 ml of 0.1 M acetic acid for 1 h, and counted by Cerenkov radiation. ERK and p38 MAPK activities were measured by immunobloting (see below) by using both (Thr202/Tyr204) and (Thr180/Tyr182) phospho-specific antibodies, respectively.

Kinetics of JNK relaxation. Confluent cell cultures were activated by increasing the NaCl concentration of the medium (by 300 mosmol/kgH2O for the control cells and by 800 mosmol/kgH2O for the adapted cells). After 15 min they were returned to the basal medium (300 and 600 mosmol/kgH2O, respectively). Cells were harvested at different time points, and JNK activity was measured. Data were analyzed by using GraphPad Prism version 3 from GraphPad software.

Assay of myo-inositol-Na cotransporter. Myo-inositol uptake was measured as previously described (25, 27) 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) 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 (3).

Measurement of Na-K-ATPase activity. Cells were harvested by trypsinolysis, washed three times in 0.25 M sucrose (10 mM Tris · HCl, pH 7.5), homogenized in a glass/Teflon homogenizer at 500 rpm, and frozen. ATPase measurement was modified from Xie et al. (28). Briefly, membranes were permeated by incubation for 10 min at room temperature with alamethicin, at a mass-to-protein ratio of 1:4. In a final volume of 300 µl, the reaction mixture contained (in mM) 3 MgCl2, 100 NaCl, 5 KCl, 1 EGTA, 5 NaN3, 50 Tris · HCl, pH 7.5, 5 [gamma -32P]ATP (0.2 mCi/mmol), and 1 ouabain as well as 100 µg of protein, when appropriate. Assays were conducted at 37°C for 0-30 min. Each reaction was terminated by addition of 1.2 ml of 0.5 M HClO4, 3.5% tungstosilicic acid. Released phosphate was converted to phosphomolybdate by adding 3 ml of 60 mM NaMoO4, 2.25 M NaCl, and extracted with 4 ml of tert-butyl alcohol. The organic phase was transferred to counting vials, and Cerenkov radiation was measured.

Hydration state in mice. Mice weighing between 18 and 22 g were housed in metabolic cages with food and water ad libitum for at least 24 h before the start of the experiment. At that point, water was removed from the "water-restricted" group and was replaced with a 5% solution of dextrose in the "5% dextrose solution" group. Urine samples were collected at different times. Mice were euthanized by cervical dislocation, kidneys were removed, and papilla and cortex were dissected in ice-cold saline.

Sample preparation for immunoblotting. Cultured cells or excised tissues were homogenized in lysis buffer as described (26). Protein content was determined by the bicinchoninic acid protein assay (Pierce). Depending on the experiment, from 25 to 100 µg of protein per lane were loaded on 10% polyacrylamide gels, subjected to electrophoresis in a Tris-glycine-SDS buffer system and electroblotted to Immobilon-NC (Millipore). Blocking, incubation with antibodies, and washing were done as described previously (26). For Western blot analysis of the alpha 1- and beta 1-subunits of the Na-K-ATPase, from 5 to 10 µg of protein per lane were loaded on 7.5% polyacrylamide gels, the rest of the procedure was as described for MAP kinases and heat-shock proteins (HSPs). Anti Na-K-ATPase beta 1-subunit was generously provided by Dr. Steven Karlish (The Weizmann Institute of Science). Identical protein loading was assessed by using the G protein Galpha i-3 as an internal standard. Horseradish peroxidase was detected by incubation with Luminol reagent (New England Biolabs), as described by the manufacturer. Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1D Image software (Kodak Digital Science).

RNA purification and Northern blot. Cytosolic RNA was isolated from IMCD3 cells by using an RNeasy mini kit (Qiagen) as described in the corresponding handbook. RNA was separated by agarose gel electrophoresis as described (14) and transferred to nylon membrane by using the Genie blotter (Idea Scientific) as suggested by the factory.

For the alpha 1- and beta 1-mRNA of the Na-K-ATPase subunits, 1.6- and 0.6-kb probes were obtained by reverse transcription of IMCD3 RNA and amplification by PCR by using the following primers: 5'-ACCATCCAAGTAATGCTGCTAACC-3' and 5'-TCAAAGCGTCCCTGAAAACG-3' for beta 1 and 5'-GCCACYATGGGGAAGGGGGTTGGAC-3' and 5'-CGGTCYAGGATCCTTTCTGG-3' for alpha 1.

Probes were radiolabeled with [alpha -32P]dCTP by random priming by using a Rediprime II kit (Amersham) and purified by spin desalting on Sephacryl S-400 HR columns.

Prehybridization and hybridization were done in rapid-hybridization buffer (Amersham) at 65°C for 2 h. Blots were washed three times in 1.5 × sodium chloride-sodium citrate-EDTA (SSPE)-0.1% SDS at room temperature and three times in 0.75 × SSPE-0.1% SDS at 65°C, exposed to X-ray film, and the results were analyzed as described above for Western blot.

Statistics. Results were analyzed by ANOVA and a Tukey-Kramer multiple comparisons test by using the InStat software package (GraphPad software). A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Development of cell lines. The stepwise increment of medium tonicity by 50 mosmol/kgH2O by addition of NaCl resulted in the development of cell lines that survived and could be passaged indefinitely in osmolalities ranging from 350 to 900 mosmol/kgH2O. This is in stark contrast to the rapid loss of cell viability observed with increasing time after exposure to 600 mosmol/kgH2O (Fig. 1). It must be noted that when urea was added to increase medium osmolality rather than NaCl, the cells did not reach confluence nor could they be maintained in culture. As shown in Fig. 2, the morphology of the adapted cells (B) is indistinguishable from that of cells living in isotonic conditions (A). Furthermore, the adapted cells had a high expression of inducible heat shock protein (C) and an enhanced rate of myo-inositol uptake (D), as would be expected from cells adapted to this environment. The apparent Michaelis-Menten constant (Km) for myo-inositol was determined to be 25 ± 4 µM (data not shown).


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Fig. 1.   Inner medullary collecting duct (IMCD) 3 and M1 cells were grown in 24-well plates to confluence and then challenged by addition of 300 mosmol/kgH2O NaCl to the culture medium. Cell survival was measured by the formation of formazan as described in EXPERIMENTAL PROCEDURES. Results are means ± SD of 6 determinations.



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Fig. 2.   A: phase contrast microscopy (×100) of IMCD3 cells grown in 300 mosmol/kgH2O NaCl (control). B: cells adapted to the addition of 300 mosmol/kgH2O NaCl [600 mosmol/kgH2O total (adapted)]. C: cell lysates of IMCD3 cells grown in normal 300 mosmol/kgH2O NaCl medium (control) and cell cultures adapted to 600 and 900 mosmol/kgH2O were analyzed by Western blot by using mouse anti-inducible heat shock protein (HSP70) monoclonal antibody (Stressgen SPA-810) and rat anticonstitutive HSC-70 monoclonal antibody (Stressgen SPA-815). Twenty-five micrograms of cell lysate protein were loaded into each lane, and experiments were performed in triplicate. D: myo-inositol uptake was measured as described by using 1.4 µM myo-inositol. The correction for specific uptake used methoxyinulin as a known nonpenetrator standard. Bars represent the mean of 6 individual determinations.

Effect of long-term adaptation to hypertonicity on MAP kinase protein and activity. Figure 3 depicts the levels of the three components of the MAP kinase family and some of the isoforms. The level of protein expression of JNK1 (A), JNK2 (B), ERK (C) and p38 MAP kinase (D) measured in three experiments was indistinguishable between the cells adapted to live at 600 mosmol/kgH2O compared with those in isotonic conditions. All three of these MAP kinases are acutely activated by hyperosmolality. Figure 4 depicts the basal activity of two of them. Neither JNK nor p38 MAP kinase basal activities were increased in cells living chronically in these conditions. We then investigated the effect of further acute increments in tonicity on the activities of JNK and p38 MAP kinase in these cells. Compared with the marked and brisk response observed in cells living at 300 mosmol/kgH2O when their bathing tonicity is increased to 600 mosmol/kgH2O, a comparable increase in tonicity produces a blunted activation of these kinases in the adapted cells. Even when media osmolality was doubled from 600 to 1,200 mosmol/kgH2O, the activity was lower than that in controls. When the linear response component of the JNK activity was quantitated, the slope of the control (0.628 ± 0.072, n = 6) was significantly different (P < 0.0002) from that of the adapted cells (0.221 ± 0.018, n = 6). Similarly, the slope of the p38 MAP kinase response in control cells was (22.72 ± 3.26, n = 3), significantly different (P < 0.02) from that of the adapted cells (4.64 ± 0.52, n = 3). A possible explanation for this observation is that despite the fact that the amount of protein was not changed, these adapted cells could not maximally activate the kinases. However, both the exposure to ultraviolet (UV) light and the acute return of the media osmolality of 300 mosmol/kgH2O (data not shown) led to robust and full activation of JNK kinase to levels comparable to those displayed by control cells in response to acute hypertonicity. A possible alternative explanation for the observed blunted response is that the adapted cells have enhanced phosphatase activity. To assess this possibility we studied the kinetics of JNK inactivation after hypertonicity-induced activation. Figure 5 shows that the rate at which JNK activity diminishes after restoration to basal conditions is very comparable in adapted and control cells. The half-time was 13.35 ± 1.42 min for control cells and 13.13 ± 0.92 min in adapted cells, not significantly different.


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Fig. 3.   Cell lysates were prepared from IMCD3 cells grown in 300 mosmol/kgH2O (control) and adapted to 600 mosmol/kgH2O. Twenty-five micrograms of lysate protein were subjected to Western blot analysis by using mouse anti-c-Jun NH2-terminal kinase 1 (JNK1), F-3 (Santa Cruz SC-1648; A), mouse anti-JNK2, D-2 (Santa Cruz SC-7345; B), mouse anti-extracellular signal-regulated kinase 1 (ERK1), C-16 and ERK2, C-14 (Santa Cruz SC-93 and SC-154; C), and mouse anti-p38 mitogen-activated protein (MAP) kinase (New England Biolabs 9212; D). Lanes represent separate independent experiments.



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Fig. 4.   IMCD3 control and cells adapted to 600 mosmol/kgH2O were grown to confluence and challenged for 15 min by addition of sterile NaCl or water to obtain discrete osmotic pressure values. A: cell lysates were then prepared for Jun kinase activity analysis. B: to measure the phosphorylated form of p38 MAP kinase, 30 µg of cell lysate protein were analyzed by Western blot by using anti-phospho-p38-MAP kinase (Thr180/Tyr182) antibody (NEB 9211), and the bands were quantitated.



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Fig. 5.   Deactivation of Jun kinase. IMCD3 control cells and cells adapted to 600 mosmol/kgH2O were grown to confluence, challenged by addition of 300 and 800 mosmol/kgH2O NaCl, respectively, for 15 min, and then returned to fresh medium. Cell cultures were harvested at increasing incubation times and assayed for Jun kinase activity. Data were normalized to the maximum value for comparison purposes. Data were subjected to best fit analysis and determined to match one-component exponential decay kinetics.

Effect of long-term adaptation to hypertonicity on Na-K-ATPase message, protein expression, and enzyme activity. Figure 6A depicts a Northern blot for Na-K-ATPase message. Densitometry measurements reveal a three to-fourfold increase in message for the protein, reflecting that the previous upregulation of the gene in acute settings (18, 30) persists in these cells. To ascertain whether this effect on messages is also reflected on protein, expression antibodies to the alpha 1- and beta 1-subunits of the protein were obtained (Fig. 6B). Adapted cells consistently had a fivefold increase in the amount of the alpha 1-subunit (n = 6) and a fourfold increase in the beta 1-subunit (n = 6). We then assessed whether the activity of the enzyme is altered in the adapted cells. Figure 6C depicts that, in fact, not only is the amount of Na-K-ATPase increased but also the activity is higher than in cells habitating in isotonic conditions.


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Fig. 6.   A: cytosolic RNA was isolated from IMCD3 control and adapted cells as described in EXPERIMENTAL PROCEDURES. Fifteen-microgram aliquots of RNA were run in duplicate agarose gels and stained with ethidium bromide reflecting equal loading (top) or transferred to a nylon membrane and probed with a 32P-labeled 1.6-kb fragment mapping to the 5' end of alpha 1-ATPase mRNA (bottom). B: cell lysates from IMCD3 control and adapted cells were analyzed by Western blot by using alpha 1-subunit ATPase (Upstate Biotechnology, no. 05-369) or beta 1-subunit ATPase antibodies. C: Na-K-ATPase activity was measured in IMCD3 control and adapted (600 mosmol/kgH2O) cell lysates.

Effect of replacement of urea for NaCl on Na-K-ATPase expression and cell viability. We attempted to assess whether the above changes in the Na-K-ATPase system were vital to the ability of the cells to adapt to hypertonicity. Because pharmacological inhibition leads to loss of cell viability under both isotonic and hypertonic conditions, we sought an alternative approach. In view of our failure to develop hypertonicity-adapted cell lines with urea and the lack of effect of this solute on Na-K-ATPase message (30), we replaced the NaCl for urea in the cells previously adapted to either 600 or 900 mosmol/kgH2O, while maintaining osmolality unchanged. The effect of this maneuver after 3 days on the expression of Na-K-ATPase and the corresponding cell viability is depicted in Fig. 7. It is of note that the replacement of NaCl for urea in cells adapted to 600 mosmol/kgH2O was associated with a downregulation of Na-K-ATPase and parallel loss of cell viability. This was not an effect of 300 mM urea per se as is reflected by the fact that when a similar maneuver was undertaken in the cells adapted to 900 mosmol/kgH2O, the Na-K-ATPase levels remained elevated and the cells remained viable. As a corollary, this observation also confirms that the acute removal of 300 mM NaCl is not responsible for the observed effect on Na-K-ATPase and cell survival seen in the cells adapted to 600 mosmol/kgH2O when 300 mM NaCl was removed and replaced with urea.


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Fig. 7.   Effects of exposure of IMCD3 cells with various solutes on alpha 1-ATPase and cell survival after 3 days. Cell lysates were analyzed for alpha 1-ATPase by Western blot, and cell survival was measured by formazan production (n = 4).

Measurements of HSP70 and Na-K-ATPase expression in M1 cells exposed to hypertonicity. In view of the high sensitivity of M1 cells to hypertonicity (see Fig. 1), we investigated whether Na-K-ATPase expression was altered in these cells. As is shown in Fig. 8, the decreased survival of the cells was associated with a failure to observe enhanced Na-K-ATPase levels. It is of interest that the cells failed to survive despite the fact that the expression of HSP70, a chaperone protein that is also implicated in the response to hypertonicity (17), was considerably elevated in this setting.


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Fig. 8.   Effect of 16 h of hypertonicity on Na-K-ATPase and HSP70 protein levels in M1 cells. Left: quantitation of 6 different experiments. Right: a representative Western blot. M1 cells were grown to confluence and challenged with 300 mosmol/kgH2O of NaCl. After 16 h, cell lysates were prepared and aliquots of 25 µg of protein were analyzed by Western blot by using mouse anti-inducible HSP70 monoclonal antibody and anti-alpha 1- ATPase. Note that the upregulation of HSP70 but not Na-K-ATPase in these cells is highly sensitive to hypertonicity.

Effect of varying states of hydration of the expression of Na-K-ATPase in the inner medulla of mice. To determine whether our observations in the cultured cells were seen in vivo, we studied the expression of Na-K-ATPase in the cortical and medullary tissues of three mice on ad libitum water intake, three mice that were further water deprived, and three mice given 5% dextrose in water to drink for 36 h. The urinary osmolality of these animals was 2,860, 4,380, and 550 mosmol/kgH2O, respectively. As is depicted in Fig. 9, the animals excreting a markedly hypertonic urine (ad libitum water and water deprived) had a greater expression of Na-K-ATPase (~30%) than animals whose urine osmolality was significantly reduced by high-water intake (P < 0.02, n = 6). No such changes were seen in the isotonic cortex.


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Fig. 9.   Effects of water hydration in mice on alpha 1-ATPase in kidney papilla and cortex tissues. Mice tissues were harvested after 36 h, and homogenates were analyzed by Western blot (n = 6). The expression of Na-K-ATPase was significantly (P = < 0.02) decreased in the papilla of water diuresis mice drinking 5% dextrose in water (D5W; top). A representative Western blot of the papilla alpha 1-Na-K- ATPase obtained from the 3 groups of mice is shown (bottom).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The survival of renal cells in the inhospitable hypertonic environment of the inner medulla of the mammalian kidney is critically dependent on the accumulation of organic osmolytes (4). The adaptive process has been extensively studied in a variety of renal cells in culture (6, 8), mostly by observing the effects of elevations in media tonicity, from 300 to 500 mosmol/kgH2O, over a brief period. The exposure of more osmotically tolerant cultured cells to higher osmolalities results in very low cell survival even after only 24 h (21). In fact, as our own data show, the percentage of cells that remain viable at 600 mosmol/kgH2O decreases rapidly with time (Fig. 1). This occurs despite the presence of 70 µM inositol in the culture media, a concentration well in excess of the Km of the sodium-inositol cotransporter of ~25 µM. In the in vivo setting, medullary renal cells are rarely in an isotonic environment and most often are surrounded by interstitial tonicities that exceed 500 mosmol/kgH2O. Thus to study the adaptive changes that these cells undergo during prolonged exposure to hypertonicity, we employed a modification of the method described by Rauchman et al. (19) and established a cell line of IMCD3 cells adapted to survive and to be passaged at osmolalities of 900 and 600 mosmol/kgH2O. These cells displayed normal-appearing morphology. Also, in agreement with the observation that adapted cells showed considerable inositol accumulation (19), we found enhanced uptake of this osmolyte. Furthermore, the expression of inducible HSP70, normally not observed in isotonic conditions but induced by acute increments in tonicity, is highly upregulated in these adapted cells.

With this background, we investigated the three members of the MAP kinase family in these cells. The activation of these MAP kinases in response to hypertonicity has now been reported by many investigators (1, 9, 22, 31). It has been established that the stimulation of the ERK pathway is not linked to the enhanced inositol uptake (1) or the transcription of the betaine and butyric acid cotransporter gene (11). Some studies have attempted to establish a role for JNK activation in survival under hypertonic conditions, although the mechanism whereby this occurs remained undefined (27). Likewise, the activation of p38 MAP kinase has been linked to the transcriptional regulation of the betaine transporter in MDCK cells (22), possibly mediated in HepG2 cells by the ORE (16). In contrast, in papillary cells, Kultz et al. (10) found that p38 MAP kinase activity is not necessary for transcriptional regulation of aldose reductase through the ORE. A systematic study of these MAP kinases in our adapted cells reveals that neither the expression nor the basal activity of the three members of the MAP kinase family is altered in such cells compared with control cells living at 300 mosmol/kgH2O (Figs. 3 and 4). Furthermore, when the cells were subjected to further increments in tonicity, the activation of both JUN and p38 MAP kinase was significantly blunted. The cells' intrinsic ability to activate the kinases is not impaired because the acute decrement of tonicity from 600 to 300 mosmol/kgH2O caused a prompt, robust response, as did the exposure to UV light.

We also explored the possibility that enhanced activity of a phosphatase could deactivate the kinase. However, the rate at which JNK activity decreased in the adapted and control cell lines, after restoration to the basal osmolality at which the cells had been living, produced very comparable deactivation curves, making it highly unlikely that phosphatase activity was significantly different. It must also be noted that because the shrinkage of the cells that accompanies acute increases in tonicity is a likely mediator of the kinase response, we increased osmolality not only by a similar absolute degree (300 mosmol/kgH2O) but also by a relative degree, doubling the osmolality to 1,200 mosmol/kgH2O in the adapted cells. Even at this level, the activity of the kinase was decreased. Because the stimulus that is responsible for the activation of the kinases in the first place is not entirely defined, the cause for this blunted response is speculative.

Our studies therefore suggest that whatever the biological role of these osmotic activated kinases may be, their role is limited to the initiation of the osmotic response, and as such may represent a nonspecific stress response, not unlike that to UV light (12). The maintenance of the adaptive response does not require either the upregulation of these proteins or their ongoing activation.

Several investigators have reported the effects of increments in osmolality on Na-K-ATPase in a variety of cultured renal cells (2, 18, 30). These studies uniformly report an upregulation of the message for both the alpha - and beta -subunits of Na-K-ATPase in response to exposures to osmolalities of 500 mosmol/kgH2O for 1 (2) and up to 24 h (18, 30). Our results reveal that the upregulation of the message is sustained beyond the early phase and is present in cells living in a high-osmolality environment. We further ascertained, as had not been previously done in renal cells, that this upregulation of message is translated into increased expression of the proteins for both the alpha - and beta -subunits of Na-K-ATPase. Furthermore, the cells displayed a marked increase in the activity of the enzyme. A similar enhancement of Na-K-ATPase activity has been observed in vascular smooth muscle cells exposed to mannitol for 48 h (15) but not previously in renal cells.

The possible significance of this upregulation of Na-K-ATPase vis a vis cell survival in hypertonicity is somewhat more difficult to assess. Pharmacological inhibition with ouabain is highly toxic under both isotonic and hypertonic conditions, and molecular transfectants have not been described. We did observe, however, that in contrast to NaCl, the addition of urea as a solute was not sufficient to allow for the production of cell lines adapted to live in hypertonicity. This is reminiscent of the effect seen by the addition of 50 mM urea to PAP-HT25 cells (29), an effect that can be counteracted by betaine. Urea also does not stimulate Na-K-ATPase production. We thus studied the effect of replacement of 300 mosmol/kgH2O of NaCl for urea in cells adapted to live at 600 and 900 mosmol/kgH2O, respectively. The replacement of 300 mosmol/kgH2O of NaCl for urea resulted in a decrease in Na-K-ATPase expression and a loss of viability of the cultured cell lines. This was not a consequence of the addition of urea per se because, when a similar maneuver was employed in cells living at 900 mosmol/kgH2O, no such loss of viability of Na-K-ATPase expression occurred. The results (shown in Fig. 7) support the need for a persistent elevation in Na-K-ATPase protein in the adaptive process. It must be noted, however, that other proteins or protective factors may have also decreased on replacement of urea for NaCl. In fact, this was true for myo-inositol uptake as well as HSP70. The latter has been particularly implicated in cell survival in the presence of urea (17). Thus the cellular response to hypertonicity is multifactorial, involving many pathways of which Na-K-ATPase is an important component. It is of interest that the very sensitive M1 cell line (Fig. 1) has low survival despite strong upregulation of HSP70, at a time when Na-K-ATPase expression remains unchanged (Fig. 8). The upregulation of Na-K-ATPase could be best described as necessary but not sufficient for cell survival under hypertonic conditions.

The mechanism whereby hypertonicity upregulates Na-K-ATPase is not fully defined. It is well known that an increase in intracellular sodium increases Na-K-ATPase message and activity (20, 24), and acute exposure to hyperosmotic media raises cell Na (7), as a number of transporters and exchangers contribute to cell volume regulation in this setting (23). The mechanisms responsible for the persistent upregulation of Na-K-ATPase in the steady-state chronic setting will require further investigation. Cell volume is restored by the persistent enhanced uptake of organic osmolytes (see Fig. 2). Many of these involve sodium-coupled transporters, and therefore the bilateral exit of sodium may be critical to maintain cellular sodium and the transmembrane electrochemical gradient that drives these transports. Presumably, when urea replaces NaCl, the uptake of osmolytes is not stimulated, and with it Na-K-ATPase is downregulated. Under such conditions, the cell line cannot be maintained.

Another aspect of our studies was the investigation of the Na-K-ATPase in the in vivo setting. The studies complement our observations in cultured cells and lend further significance to them. Mice, as well as other mammals, excrete a concentrated urine in the basal state. As such, the expression of Na-K-ATPase in animals that were water deprived for 36 h, and whose osmolality was only marginally higher than that in the water ad libitum group, was not significantly higher. However, both groups of animals had a much higher expression of Na-K-ATPase than occurred when mice were given 5% dextrose in water to drink and whose osmolality was only 550 mosmol/kgH2O. This observation suggests that in the in vivo the medullary Na-K-ATPase is tonically stimulated to maximal expression and is downregulated during urinary dilution.

In summary, we successfully developed cell lines of IMCD cells that survive long term in hypertonic media. Our experiments are the first to reveal that the MAP kinase pathways are neither upregulated nor activated in these conditions, and in fact their activation in the face of further increments in tonicity is blunted, suggesting a change of sensitivity to hypertonicity. These kinases may play a critical role in the generation of cellular events that allow for survival in hypertonic conditions but are clearly not involved in the maintenance of those events. In contrast, we have shown for the first time that the increased expression and activity of Na-K-ATPase are sustained in such cells, as well as in medullary tissues of rodents excreting hypertonic urine. The upregulation of the protein is most likely an important component of a much larger number of adaptive processes that allow the cells to survive in those conditions. Decrement in the expression of the protein appears to be associated with loss of cell variability and of the cultured cell line, suggesting a vital role for this protein in the adaptive process.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19928.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Berl, Division of Nephrology, Dept. of Medicine, Univ. of Colorado School of Medicine, Denver, CO 80262 (Email: Tomas.Berl{at}uchsc.edu).

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

Received 31 August 2000; accepted in final form 8 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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