1 Department of Medicine, Brigham and Women's Hospital, Harvard Institutes of Medicine, Boston 02115; 2 Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655; and 3 Department of Surgery, University of California, San Francisco, California 94143
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ABSTRACT |
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Renal medullary cells are exposed to elevated and variable osmolarities and low oxygen tension. Despite the harsh environment, these cells are resistant to the effects of many harmful events. To test the hypothesis that this resistance is a consequence of these cells developing a stress tolerance phenotype to survive in this milieu, we created osmotically tolerant cells [hypertonic (HT) cells] by gradually adapting murine inner medullary collecting duct 3 cells to hyperosmotic medium containing NaCl and urea. HT cells have a reduced DNA synthesis rate, with the majority of cells arrested in the G0/G1 phase of the cell cycle, and show constitutive expression of heat shock protein 70 that is proportional to the degree of hyperosmolarity. Unlike acute hyperosmolarity, chronic hyperosmolarity failed to activate MAPKs. Moreover, HT cells acquired protein translational tolerance to further stress treatment, suggesting that HT cells have an osmotolerant phenotype that is analogous to thermotolerance but is a permanent condition. In addition to osmotic shock, HT cells were more resistant to heat, H2O2, cyclosporin, and apoptotic inducers, compared with isotonic murine inner medullary duct 3 cells, but less resistant to amphotericin B and cadmium. HT cells demonstrate that in renal medullary cells, hyperosmotic stress activates biological processes that confer cross-tolerance to other stressful conditions.
heat shock protein 70; nephrotoxins; thermotolerance; cell cycle; inner medullary collecting duct 3
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INTRODUCTION |
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CONCENTRATED URINE RESULTS from the operation of the countercurrent system, and accumulation of high amounts of NaCl and urea in the medullary interstitium is an important element of this process. As a consequence, cells in the renal medulla must deal with constantly changing extracellular solute concentrations ranging from 300 to 1,200 osM in humans. In addition to this osmotic stress, the countercurrent system results in a reduced oxygen tension in the medulla, confronting cells in this kidney zone with potential hypoxic episodes (1). To survive and function in such a harsh environment, renal inner medullary cells developed a specialized program that enables them to adapt to this milieu. This program involves the activation of signal transduction kinases, synthesis of stress proteins, and accumulation of compatible organic osmolytes, responsible for the maintenance of cell volume and intracellular ionic strength (2, 11, 33).
In addition to withstanding hyperosmolarity, those cells appear resistant to ischemia-reperfusion episodes and many nephrotoxins by mechanisms still unknown (14). Ischemia-reperfusion injury is a proximal tubule lesion that has little impact on renal inner medullary cells (32). Most heavy metals, including cadmium, lead, and mercury, adversely affect the proximal tubules but not the collecting ducts (9). These observed differential sensitivities to injury led us to hypothesize that the physiological adaptation to hyperosmolarity might confer enhanced tolerance of renal medulla cells to different types of injury, with the induction and constitutive expression of stress-responsive genes.
In contrast to the osmotolerant phenotype of renal medullary cells observed in vivo, osmotic stress in vitro has a lethal effect on kidney cells. Exposure of murine inner medullary collecting duct 3 (mIMCD-3) cells, derived from the mIMCD (26), to hyperosmolar NaCl or urea causes a dramatic decrease in cell viability (29). Cell death is associated with suppression or disruption of many cellular processes, including RNA, DNA, protein synthesis, and cell division (28). In contrast, mIMCD-3 and Madin-Darby canine kidney cells exposed to an equimolar combination of NaCl and urea are significantly more osmotolerant (21, 29, 39), indicating that NaCl and urea together activate a survival program conferring enhanced osmotolerance.
Herein, we characterize a physiological mechanism of stress tolerance with a collecting duct cell line (mIMCD-3 cells) adapted to hyperosmolar conditions by a combination of NaCl and urea mimicking the osmotic milieu in the renal medulla. We have termed these hypertonic (HT) cells. In them, biosynthesis rates are slowed and the stress-inducible form of heat shock protein 70 (HSP70) is expressed constitutively. Furthermore, HT cells exhibit a greater resistance to many different nephrotoxins. These results with the HT cell lines indicate that the hyperosmotic stress faced by the renal medulla activates biological mechanisms that result in cells exhibiting cross-tolerance to other stressful conditions.
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MATERIALS AND METHODS |
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Cell culture and viability assays. mIMCD-3 cells were grown in DMEM/F-12 plus 10% FBS and 2% penicillin-streptomycin. For adaptation, mIMCD-3 cells were grown in 10 ml culture medium, in which osmolarity was increased ~100 mosM every 48 h with the addition of 1 ml of a stock solution containing equimolar concentrations of NaCl and urea. At least three different experiments with six replicates were performed between cell passages 15 and 52 to obtain the multiple observations. During this period of time, adapted cells maintained all their characteristics, and we have termed these stable lines, at the highest osmolarity (1,270 mosM), HT cells. To assess viability, a crystal violet assay was used. As described before (29), cells were seeded at 104 cells/well in 96-well flat-bottom plates, incubated, and exposed to different treatments. After treatment, DNA of adherent cells was stained with crystal violet. The viability percentage of treated cells was defined as the absorbance relative to untreated cells. The percentage of cytotoxicity was defined as 100% (untreated cells) minus the percent viable cells. To confirm viability results, cytotoxicity was also monitored by light microscopic evaluation of both the supernatant and the adherent cells using trypan blue viability assay.
[3H]Thymidine, [3H]uridine, and [3H]leucine incorporation. Cells were seeded in 96-well plates at 5 × 105 cells/well and grown to subconfluence. To measure rates of DNA, RNA, or protein synthesis, cells received a pulse of labeled substrate (New England Nuclear) as follows: 0.5 µCi/ml [3H]thymidine, 1 µCi/ml [3H]uridine, or 0.5 µCi/ml [3H]leucine. After 6 h of exposure to uridine or leucine, or 12 h to thymidine, cells were trypsinized, washed, and harvested by using a 1205 Betaplate System (Wallac, Finland). Incorporation rates were obtained by scintillation counting in the presence of Betaplate Scint.
Northern blot analysis. As described earlier (29), cells were washed and total RNA was isolated by using the RNAzol B method (Tel-Test). Total RNA (10 µg) was fractionated and transferred overnight to a nylon membrane. For probes, human cDNA fragments of HSP70 and BiP (both from the American Type Culture Collection) were labeled with [32P]dCTP using a random hexamer labeling kit (Pharmacia). Blots were prewashed and then hybridized overnight at 42°C in 40% formamide, 10% dextran sulfate, 7 mM Tris (pH 7.6), 4× SSC (1× SSC contains 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0), 0.8× Denhardt's solution (1× Denhardt's solution consisted of 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 20 µg/ml salmon sperm DNA, 0.5% SDS, and the 32P-labeled cDNA probes (106 cpm/ml). Next, blots were washed at room temperature (2× SSC and 0.1% SDS for 20 min) and then at 50°C (0.2× SSC and 0.1% SDS for 20 min). The HSP70 probe detected both inducible (HSP70) and constitutive (heat shock cognate protein 70) transcripts. Autoradiography was performed with Reflection TM (NEN Research Products) film and an intensifying screen.
Preparation of cell lysates and Western blot analysis. As previously described (27), cells were washed with ice-cold PBS at correspondent osmolarity and harvested in 1% Triton, 50 mM Tris (pH 7.5), and 1 mM DTT. Protein concentration was determined by Bradford assay (Bio-Rad). Equal amounts (40 µg) of total protein from the cell lysates were resolved on 10% SDS-PAGE and transferred to nitrocellulose (Nitropure, MSD). Membranes were probed with a monoclonal antibody against inducible HSP70 or HSP72 (1:1,000; SPA-810, StressGen). Detection was performed with the ECL system (Amersham). Band intensity was quantitated densitometrically (GS-700 Imaging Densitometer and software).
Casein-affinity chromatography.
For chaperone purification, the experiment was performed essentially as
described (27) with slight modifications. A 2-ml column of
casein conjugated to cyanogen bromide-activated Sepharose (Sigma) was
unfolded by incubation with 6 M urea and 1 M -mercaptoethanol. The
affinity column was washed and 5 mg of cellular protein (from cell
lysates) were applied to the column. The following fractions were
obtained and analyzed by SDS-PAGE: 1) flow-through,
unbounded proteins; 2) Mg-wash, wash proteins unspecifically
trapped in the column (last 1.5 ml of the 5-ml wash, an indication of
the wash effectiveness); 3) ATP elution, for putative
chaperones elution; and 4) acid elution, for proteins
remaining on the column after the ATP elution.
Preparation of cell lysates and immunoblot analysis to detect intracellular signaling molecules. Cell lysate preparation and immunoblot analysis were performed essentially as described (38). Briefly, after appropriate solute treatment, cells were washed with ice-cold PBS of equal osmolarity and lysed in 200 µl of protein lysis buffer containing 50 mM Tris(hydroximethyl)aminomethane (Tris) (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium orthovanadate, 100 mM sodium fluoride, 10 µg/ml leupeptin, 20 µg/ml antipain, 100 µg/ml benzamidine, and 10 µg/ml aprotinin. Cell lysates were clarified at 15,000 g for 10 min at 4°C, and protein content was determined by using the Bradford microassay (Bio-Rad). Equal amounts of protein (80 mg) were boiled in SDS-Laemmli sample buffer, resolved in 12% SDS-PAGE, and transferred to polyvinylidene difluoride (Immobilon P, Millipore). Membranes were probed with antiactive MAPK polyclonal antibody (1:5,000; Promega), which recognizes the phosphorylated active form of ERK1 and ERK2; antiactive JNK polyclonal antibody (1:2,000; Promega), which recognizes the phosphorylated active form of the JNK isoforms; and phosphospecific p38 MAPK antibody (1:2,000; New England Biolabs), which recognizes the phosphorylated active form of p38 MAPK.
Two-dimensional SDS-PAGE. Two-dimensional SDS-PAGE was performed as described previously (34). Briefly, cells were labeled with [35S]methionine (5 µCi) for 12 h in medium lacking methionine and supplemented with 10% FBS. In addition, cells were submitted to heat shock treatment (43°C/1 h) and then returned to 37°C and labeled for 12 h with [35S]methionine. Cells were harvested in 0.1% Triton X-100, and equal amounts of lysates were loaded onto pH 5-7 isoelectric-focusing gel. After isoelectric focusing, the proteins were resolved by 12.5% SDS-PAGE. Gels were fluorographed and exposed to film.
Flow cytometry and cell cycle analysis. Staining was performed according to the Dana Farber Core Flow Cytometry Center protocol. Briefly, cells were cultured in 60-mm plates until subconfluence, trypsinized, washed with ice-cold PBS (without divalent cations), and suspended in ice-cold PBS to a concentration of 2 × 106 cells/ml. One milliliter of cell suspension was vortexed while 1 ml of ice-cold 80% ethanol was added in a drop-wise fashion. For fixation, cells were incubated for 30 min on ice. Fixed cells were washed and raised in 1 ml of PBS containing propidium iodide (2.5 µg/ml) and RNase (50 µg/ml). Cells were incubated for 30 min at 37°C in the dark. Subsequently, the material was submitted to flow cytometric analysis of DNA content and cell cycle progression (Scalibur Scan, Becton-Dickinson, and CellFit Software).
Immunostaining.
Cells were grown on sterile glass slides (TechMate Blue Capillary Gap
Micro Slides, Ventana Medical Systems) in 100-mm petri dishes. After
confluence, cells were washed twice with PBS and fixed with ice-cold
methanol for 30 min, air dried for 20 s, and stored at
70°C. Kidneys were removed from adult male Sprague-Dawley rats (Charles River Labs) and frozen on dry ice. Twelve-micrometer frozen sections were cut with a Leica CM3000 cryostat and thaw mounted
on Superfrost Plus slides (Fisher Scientific). The immunostaining was
performed as described previously (27). Cultured cells and kidney sections were photographed with a ×100 oil-immersion objective (SPlan 100) mounted on an Olympus BH-2 microscope using Ektachrome 100 color-slide film.
Statistical analyses. Statistical analyses were performed with StatView Software (Abacus Concepts) by using a t-test and Fisher's paired least-significant difference multiple comparisons test when appropriate. Data are expressed as means ± SE, and significance was assigned to P < 0.05.
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RESULTS |
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Establishment of adapted cells. mIMCD-3 cells were exposed to culture medium containing gradually increased amounts of NaCl and urea in equimolar concentrations. Cells were adapted to a range of osmolarities with the highest osmolarity achieved being 1,270 mosM. These cells survived and retained their viability at high osmolarity even after storage in liquid nitrogen. We have termed these stably adapted lines (1,270 mosM) HT cells.
Altered biosynthesis and growth properties of HT cells.
RNA and protein synthesis rates were measured in cells adapted to a
range of osmolarities (Fig.
1A). With increasing medium osmolarity, cells showed a progressive decline in RNA synthesis, reaching a minimum of 35% of the control level at 1,270 mosM. In
contrast, changes in the rate of protein synthesis showed a biphasic
behavior, increasing by up to 65% above control levels when adapted to
460 and 610 mosM and decreasing below control levels when medium
osmolarity was 760 mosM or higher. At the highest level of osmolarity
(1,270 mosM), the rates of both protein and RNA synthesis were lowest.
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Overexpression of HSP70 in HT cells.
In renal epithelial cells, acute hyperosmolarity induces the expression
of HSP70 (2, 29). As shown in Fig.
2, A and B, adapted
cells exhibited a robust expression of inducible HSP70 mRNA (up to 28×
increase) and protein. Of note, the HSP70 protein content increased in
correlation with rising medium osmolarity, with highest expression
levels detectable at highest osmolarity. Constitutive HSP70 (heat shock
cognate protein 70) mRNA was increased 1.5 times compared with the
control.
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MAPKs are not activated in HT cells. In renal epithelial cells, acute hyperosmolar stress, elicited by either urea or NaCl, activates the three principal MAPK cascades, including the ERKs, JNKs, and p38 MAPK (33). To evaluate the activation status of MAPK in HT cells, Western blot analyses were performed by using an antibody against the active phosphorylated forms of ERK, JNK, and p38 MAPK. In isotonic mIMCD-3 cells, we did not observe any significant MAPK activity (Fig. 2D, Isotonic medium), whereas all three MAPKs (ERK, JNK, and p38 MAPK) responded strongly to an acute addition of 100 mM NaCl. Of note, in the HT cells no constitutive MAPK activity was observed. However, these kinases became activated when HT cells were acutely exposed to additional stress, for example, changing cell culture medium (data not shown). Thus in the chronic hyperosmolar state, constitutive MAPK activation does not appear to be required to maintain the altered phenotype.
Subcellular localization of HSP70.
After heat shock treatment, newly synthesized HSP70 protein is known to
rapidly localize within the nucleus and, in particular, the nucleolus
(35). Because we also found high levels of HSP70 expression in HT cells, we examined its localization in them. HSP70
immunostaining (Fig. 3) revealed little
or no staining in mIMCD-3 cells maintained under isotonic conditions
(310 mosM), a result consistent with our Western blot analysis results
(Fig. 2B). With increasing osmolarity, the cells showed a
proportional increase in HSP70 immunostaining (Fig. 3A),
with the protein being present in both the cytoplasm and the nucleus
but not the nucleolus (Fig. 3B). Even under the conditions
of highest osmolarity, no nucleolar staining was observed (Fig.
3B, 1,270 mosM). In contrast, heat shock treatment of
isotonic mIMCD-3 cells resulted in robust nucleolar immunostaining for
HSP70 (Fig. 3B, Heat). From this last observation, we infer
that osmotic stress, unlike thermal stress, has little or no adverse
effect on the integrity of maturing ribosomes within the nucleolar
compartment.
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Localization of HSP70 within the intact kidney.
Osmolarity-dependent variation in the expression of HSP70 similar to
that seen in adapted cells was also observed in the intact kidney (Fig.
4). HSP70 immunostaining was largely
confined to the collecting ducts in all regions of the kidney. The
intensity of staining and hence HSP70 levels paralleled the
corticomedullary osmotic gradient and were lowest in the cortex (Fig.
4A), intermediate in the outer medulla (Fig. 4B),
and highest in the inner medulla (Fig. 4C). The only other
HSP70 staining observed was in the urothelial lining of the collecting
system (Fig. 4C). Thus osmolarity-dependent constitutive
expression of HSP70, under isovolemic conditions, is also a
property of renal medullary cells in vivo.
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Preferential expression of stress proteins in HT cells.
After heat shock treatment, preferential translation of mRNAs encoding
the HSPs along with the repression of synthesis of non-stress-related
proteins has been observed (6). Because HT cells showed
high-level expression of inducible HSP70, we examined whether the cells
might show preferential protein synthesis patterns similar to that
observed for cells subjected to heat shock treatment. Control mIMCD-3
cells and HT cells were labeled to steady state with
[35S]methionine. Cell lysates were prepared, and the
pattern of protein synthesis was examined by two-dimensional
electrophoresis. As seen in Fig. 5, the
amount and the pattern of proteins being synthesized were indeed
different in the HT cells (Fig. 5B) compared with mIMCD-3
cells (Fig. 5A). Although the gels were loaded with the same
amount of total protein, the overall content of radiolabeled proteins
appeared higher in the mIMCD-3 cells compared with the HT cells. This
observation is consistent with those presented in Fig. 1A
showing that HT cells displayed a dramatically reduced rate of overall
protein synthesis. Note as well that the general pattern of protein
synthesis was significantly different between the mIMCD-3 cells and the
HT cells. Specifically, the HT cells showed high-level expression of a
select group of proteins, including HSP70, along with a reduced
expression of other proteins that were expressed in the mIMCD-3 cells.
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HT cells exhibit enhanced stress tolerance.
In all organisms, exposure to a mild heat shock treatment elicits a
transient state of thermoresistance known as "acquired thermotolerance." Moreover, cells can exhibit "cross-tolerance"; transient exposure to one stressor can confer enhanced tolerance to
others (23). The degree of acquired tolerance correlates with the expression levels of different HSPs, particularly HSP70 (13). Therefore, we examined whether HT cells might
exhibit enhanced tolerance to other types of metabolic stress. Isotonic mIMCD-3 cells and HT cells were exposed to a severe heat shock treatment (46°C/4 h) (Fig.
6A). Assessment of
cytotoxicity 24 h after treatment clearly demonstrated that HT
cells were less heat sensitive than isotonic cells (15 vs. 56%,
P < 0.01). In addition, we analyzed whether HT cells
could tolerate acute hyperosmotic stress, a circumstance similar to
that occurring in vivo during transitions in hydration state. As shown
in Fig. 6, B-D, HT cells were more resistant
to acute addition of NaCl, urea, or a combination of NaCl and urea
compared with isotonic cells. Furthermore, the maximal osmolarities
(i.e., combination of NaCl and urea) tolerated by at least 50% of the
cells for 24 h were 2,540 mosM for HT cells but only 750 mosM for
the isotonic cells.
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DISCUSSION |
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Medullary hyperosmolarity and hypoxia are an inevitable accompaniment of efficient urinary concentration. In mammals, renal IMCDs are rather unique in their ability to live in such a harsh physiological environment. Mimicking the postnatal and progressive acquisition of urinary concentration ability in vivo (22), we created osmotically tolerant cell lines, called HT cells, by gradually adapting mIMCD-3 cells to a hyperosmotic environment up to 1,270 mosM in vitro. In contrast to the apoptotic effect of high extracellular concentrations of either NaCl or urea, a combination of both, as exists in vivo, promoted such an acclimation process (21, 28, 39).
In HT cells, DNA synthesis was reduced by 25%, suggesting a decreased rate of cell division (Fig. 1B, b). Consistent with this, HT cells exhibited an increase in the proportion of cells within the G0/G1 stage and a corresponding decrease in cells in the S and G2/M phases (Fig. 1B, a). It has been shown that during the G1 phase, cells respond to extracellular signals by either advancing toward another division or withdrawing from the cycle into a resting state (G0) (31). Thus adaptation to hyperosmolarity appears to slow proliferation by delaying the exit from the G0/G1 stage. This response may be mediated, in part, through changes in p53, as described in acute NaCl stress, determining cell growth arrest and protection from apoptosis (4).
With increasing extracellular osmolarity, the rate of RNA synthesis decreased (Fig. 1A). In contrast, the rate of protein synthesis showed a biphasic profile depending on the cell culture medium osmolarity (Fig. 1A). In cells adapted to lower osmotic conditions, protein synthesis was increased, whereas under higher osmotic conditions the cells exhibited a reduced rate of protein synthesis. These altered rates of biosynthesis undoubtedly reflect changes in gene expression but also suggest that there are two different mechanisms of adaptation depending on the severity of hyperosmotic stress.
Increased expression of HSPs and molecular chaperones is a ubiquitous feature of cells exposed to acute, but typically transient, stress conditions. In contrast, a constitutive high level of inducible HSPs (e.g., HSP70) is not well known, particularly in mammals. In a number of organisms, induced expression of HSPs can be a marker for the adaptation to cyclic environmental changes, as well as a mechanism that allows different species to live in especially harsh environments (8). We found that a progressive increase in the expression of HSP70 paralleled the adaptation of the cells to high osmotic stress (Figs. 2 and 3). A comparable relationship was also found in vivo, where levels of inducible HSP70 expression followed the corticomedullary osmotic gradient (Fig. 4).
The use of heterologous promoters to force HSP70 expression has shown that constitutively expressed HSP70 coalesces into granules, and the protein present in these granules appeared to be irreversibly inactivated. It could not be dispersed with a second heat shock, and cells containing these granules did not show thermotolerance (7). Performing experiments with a denatured protein-affinity column, we showed that HSP70 that accumulates in HT cells possesses chaperone activity. Subcellular localization of HSP70 in cells chronically exposed to osmotic stress was comparable to that observed in acutely stressed cells, with a characteristic distribution in the nucleus and the cytoplasm but not in the nucleoli (27). This similarity in distribution pattern, which appeared to be independent of the severity of the osmotic stress, most likely reflects similar subcellular roles for HSP70 in acutely and chronically stressed cells (Fig. 3B). In contrast to heat-shocked mIMCD-3 cells (Fig. 3B, Heat), the absence of nucleolar localization of HSP70 in HT cells suggests that there is no change in nucleolar activity in osmotic stress. Nevertheless, the high levels of HSP70 seen in HT cells most likely represent the reason for the increased tolerance of these cells to hyperosmotic (Figs. 6, B-D) as well as heat (Fig. 6A) stress. This hypothesis is strengthened by the observation that the degree of osmotic tolerance is proportional to the level of osmolar adaptation and consequently to the cellular content of HSP70 protein (data not shown).
In contrast to the hypertonic activation of MAPKs observed after acute treatment (33), no active MAPKs were detectable in HT cells. This, together with the observation that other acute stresses were able to elicit MAPK activity in HT cells (data not shown), suggests that MAPKs are primarily involved in the acute response to hyperosmotic stress. This in vitro observation implies that the constitutive activation of MAPKs detected in vivo in inner medullary cells (36) may be secondary to other stress factors found in the inner medulla and not related to the chronic environmental hyperosmolarity in this kidney zone. Although MAPK activation has been linked to HSP expression in response to acute hyperosmolarity (30), our data suggest that MAPKs are not crucial in the regulation of the constitutive expression of HSP70 induced by chronic hyperosmolar stress.
As a typical response to a severe heat shock, normal cellular translational activity is repressed. After heat shock, cells recover full translational activity over a relatively long period of time (6). In cells exhibiting acquired thermotolerance, exposure to a second heat shock has no effect on general protein synthesis, a phenomenon called "translational tolerance" (19). This observation suggests that translational events are key determinants of the phenomenon of thermotolerance. Evaluation of the protein expression pattern in HT cells disclosed an overall decrease in protein synthesis. Interestingly, a superimposed heat shock did not further decrease translational activity in these cells (Fig. 5), suggesting that HT cells had acquired characteristics of a translational thermotolerant cell. It has been shown that the recovery of general protein synthesis starts as soon as a certain threshold of HSP70 expression is attained (6). Therefore, the translational tolerance observed in HT cells might be related to the high levels of HSP70 expression.
For analysis of the stress-tolerant phenotype, we tested the resistance of HT cells to known nephrotoxins, including therapeutic drugs, oxidant stress, and heavy metal compounds. Those stressors have in common the capacity of inducing cell death by apoptosis (12, 16-18, 24, 25, 28). In addition, there is evidence that HSPs play a role in apoptosis, because overexpression of HSP70 can prevent apoptosis (10, 20). We showed that HT cells are more resistant to toxic levels of osmotic, heat, oxidative (H2O2), cyclosporin, and mitomycin C stress than isotonic mIMCD-3 cells, suggesting the development of a cross-tolerant phenotype to many other stressors. On the other hand, high doses of ceramide (a sphingolipid metabolite and an inducer of apoptosis), amphotericin B, or cadmium were more toxic for HT cells than isotonic cells. The cell injury induced by amphotericin B is determined by cell membrane modification and augmented inorganic ion influx (18), and the increased sensitivity of HT cells to amphotericin B in vitro correlates with the known toxicity of amphotericin B for IMCD cells in vivo (5), suggesting that this substance is only toxic to IMCD cells in combination with osmotic stress. Of note, cadmium is not toxic to IMCD cells in vivo, but this appears to be due to the induction of metallothionase enzymes in the proximal tubules, which are known to chelate and detoxify cadmium before it reaches the IMCDs (15). Heat shock is known to cross protect against heavy metals, yet we did not find this to be the case in HT cells. This discrepancy might be explained by differences we have shown here in localization and function of HSP70 in HT vs. heat-shocked cells (35). Of particular note, as observed in the cadmium-chloride cross-tolerance experiments, there was no direct correlation between HSP70 levels and cell survival.
In conclusion, the present investigation demonstrates that a physiological condition, namely, hyperosmolarity, can elicit a stress tolerant phenotype in mammalian cells. Using HT cells as a model, this cellular phenotype is characterized by reduced biosynthetic and cell-division rates, related to a slowed cell cycle progression, constitutively high levels of inducible HSP70, and translational thermotolerance, in the absence of overt activation of MAPK signaling. The antiproliferative effect, observed in association with the hyperosmolar adaptation of HT cells, as well as the high levels of HSP70, most likely represents mechanisms enabling cells to survive in the harsh renal medullary environment. Because HT cells were cross-tolerant of several stress factors other than hyperosmotic stress, one can conclude that the osmotolerant phenotype may contribute to the ability of renal medullary cells to also withstand injuries that are detrimental to other regions of the kidney and other organs. Therefore, further studies of this adaptive mechanism might open new approaches in the prevention or treatment of acute renal injury.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51606 and DK-36031. S. R. Gullans is an Established Investigator of the American Heart Association. B. C. Santos was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, 200926/94-2(NV).
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FOOTNOTES |
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Present addresses: B. C. Santos, Dept. of Medicine, Renal Div., Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-900, Brazil; and J. M. Pullman, Surgical Pathology, Moses Campus, Montefiore Medical Ctr., Bronx, NY 10467.
Address for reprint requests and other correspondence: S. R. Gullans, Brigham and Women's Hospital, Ctr. for Neurologic Disease, 65 Landsdowne St., Cambridge, MA 02139 (E-mail: sgullans{at}rics.bwh.harvard.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.
First published October 29, 2002;10.1152/ajprenal.00058.2002
Received 11 February 2002; accepted in final form 22 October 2002.
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