Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury

Bento C. Santos1, James M. Pullman2, Alejandro Chevaile1, William J. Welch3, and Steven R. Gullans1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Osmotic adaptation gradually affects RNA and protein synthesis and leads to decreased cell proliferation and cell cycle arrest. A: rates of RNA (filled bar) and protein (gray bar) synthesis in isotonic (310 mosM) and adapted murine inner medullary collecting duct 3 (mIMCD-3) cells to hyperosmolarity. Cells were pulse labeled with either [3H]uridine or [3H]leucine for 6 h to determine RNA and protein synthesis rates, respectively. Each value represents the mean ± SE of 18 observations (3 different experiments with 6 replicates), expressed as percentage of the control level. B: comparison of cell cycle distribution and proliferation rate of hypertonic (HT; 1,270 mosM) and isotonic mIMCD-3 cells. Relative numbers of cells in different phases of the cell cycle were determined by flow cytometric measurements of DNA content (a); each bar represents the mean ± SE of 4 observations. To determine proliferation rates, equal numbers of HT and control mIMCD-3 cells were pulse labeled with [3H]thymidine for 12 h (b); each bar represents the mean ± SE of 27 observations (4 different experiments with 7 replicates). *P < 0.05.

Flow cytometric analysis of cellular DNA content (Fig. 1B, a) showed that HT cells exhibit an increase in the proportion of cells in G0/G1 phase compared with isotonic cells (68 vs. 57%, P = 0.002) and a corresponding decrease in the proportion of cells in S (10 vs. 16%, P = 0.006) and G2/M phase (23 vs. 28%, P = 0.002). The cell proliferation rate (Fig. 1B, b), measured as DNA synthesis, was reduced by 25%, indicating that the altered cell cycle profile was associated with a reduced rate of cell division. Thus adaptation to hyperosmolarity appears to reduce cell proliferation rates by slowing cell cycle progression at the G0/G1 stage.

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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Fig. 2.   Heat shock protein 70 (HSP70) expression increased during the adaptation of mIMCD-3 cells to osmotic stress without MAPK activation. A: relative abundance of constitutive (HSC70) and inducible HSP70 mRNA and protein. B: representative blots showing constitutive HSP70 [heat shock cognate protein 70 (HSC70)] mRNA and inducible HSP70 mRNA and protein expression in isotonic mIMCD-3 cells (310 mosM) and cells adapted to high osmolarity. C: representative blot of proteins eluted from an unfolded casein-affinity column probed with anti-HSP70 antibody to determine the chaperone activity of HSP70 in HT cells (1,270 mosM). Protein lysates (Total) from HT cells were applied to the column. Fractions were collected before (Flow through, unbound fraction) and after sequential washing with MgCl2 (Mg wash), Mg-ATP (ATP elution), and acid (Acid elution). D: representative Western blot analysis of mIMCD-3 cells 30 min after addition of fresh isotonic medium, fresh medium containing 100 mM NaCl, or HT cells. Blots were sequentially probed with an antibody against the phosphorylated forms of ERK, JNK, and p38 MAPK.

With the use of a denatured protein-affinity column, the chaperone activity of the HSP70 expressed in HT cells was evaluated. In Fig. 2C, lane 1, the total HSP70 content in protein lysate from HT cells is shown. Application of this lysate to the unfolded casein-affinity column revealed that some of the HSP70 failed to bind to the casein and was eluted (Flow through, lane 2). When the column was washed with buffer containing MgCl2, in the absence of ATP (Mg wash), very little HSP70 was eluted, indicating the specific requirement of ATP for elution. In comparison, addition of ATP to the elution buffer (ATP elution) caused a dramatic release of HSP70. In the last step (Acid elution), an acid wash failed to elute any additional HSP70. These data demonstrate that HSP70 constitutively expressed in the HT cells exhibits the functional characteristics of a molecular chaperone.

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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Fig. 3.   Immunocytochemical localization of inducible HSP70 in cells adapted to increasing osmolarities. Osmolarity is indicated for each panel, except the last, which shows heat shocked cells (Heat). The panels labeled 310 mosM show isosmolar cells. Brown 3'-3-diaminobenzidine stain shows immunostaining for HSP70, and blue hematoxylin counterstain shows the nuclei. A: low-magnification views showing that HSP70 levels increased proportionately to the osmolarity of adaptation (magnification, ×500). B: high-magnification views (×5,000) showing that HSP70 was present in both nucleus and cytoplasm of adapted cells, but not in the nucleoli, which showed only hematoxylin counterstain. In contrast, transiently heat-shocked (42°C/30 min) isotonic mIMCD-3 cells showed localization of HSP70 in nucleoli, as well being present in the nucleus and cytoplasm.

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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Fig. 4.   Immunohistochemical localization of inducible HSP70 in sections of rat kidney. Brown 3'-3-diaminobenzidine stain shows localization of HSP70. A: cortex with minimal immunostaining. B: outer medulla with increased staining of collecting duct. C: inner medulla with strong staining of collecting ducts and urothelium of the collecting system. Magnification, ×100.

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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Fig. 5.   Two-dimensional gel electrophoresis of 35S-labeled proteins isolated from control mIMCD-3 cells or HT cells, with or without heat shock treatment. Control mIMCD-3 cells (A) or HT cells (B) were pulse labeled with [35S]methionine for 12 h. In addition, mIMCD-3 cells (C) or HT cells (D) were submitted to heat shock treatment (43°C/1 h) and then returned to 37°C and labeled for 12 h with [35S]methionine. Gels were loaded with identical amounts of protein. Downward arrow, HSC70; upward arrow, HSP70.

Two-dimensional gel analysis of mIMCD-3 cells after a 43°C/1 h heat shock treatment revealed the expected increase in inducible HSP70 expression along with the reduced expression of nonstress-related proteins in the heat shock-treated mIMCD-3 cells compared with their nonheated counterparts (Fig. 5, C vs. A, respectively). In contrast, heat shock treatment of the HT cells did not result in any major changes in the protein expression. No further increase in HSP70 expression was observed nor were there any significant effects on the expression of other proteins (Fig. 5, D and B). These observations are consistent with the idea that HT cells have adopted a stress-like phenotype characteristic of cells subjected to transient hyperthermia.

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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Fig. 6.   HT cells are more tolerant of osmotic and thermal stress. Viability of HT and control mIMCD-3 cells after acute heat stress or hyperosmotic stress was evaluated by crystal violet assay. A: mIMCD-3 cells or HT cells were exposed to severe heat shock (46°C/4 h); bars represent the mean value ± SE of 50 and 89 observations, respectively. The differences in sensitivity observed between isotonic and HT cells were statistically significant (P < 0.01). The effect of acute hyperosmolar stress was tested by exposing isotonic and HT cells 24 h to varying concentrations of NaCl (B), urea (C), or NaCl+urea (D). Each point is the mean ± SE of 17 observations. Statistical significance of differences was tested by using Fisher's paired least significant difference test for multiple comparisons (*P < 0.05).

We tested the resistance of HT cells to nephrotoxins (Fig. 7), including therapeutic drugs, oxidant stress, and heavy metal compounds. Interestingly, HT cells exhibited a very diverse sensitivity to these different noxious agents. Hydrogen peroxide (Fig. 7A), used to mimic ischemia-reperfusion injury, was considerably less cytotoxic to the HT cells than to isotonic mIMCD-3 cells. Similar stress resistance was observed after exposure to cyclosporin (Fig. 7B), with nearly twofold less cell death seen in HT cells exposed to 200 µg/ml. At all doses of mitomycin C, a DNA synthesis inhibitor used in the treatment of solid tumors, which can cause glomerular and proximal tubular but not inner medullary injuries in vivo (3) (Fig. 7C), was less cytotoxic to HT cells than to isotonic control cells. Ceramide (Fig. 7D), an inducer of apoptosis implicated in renal ischemia-reperfusion injury (37), was slightly less toxic to HT cells at a lower dose (25 µM) but equally toxic to both cell types at higher doses (50 and 100 µM). In contrast to the enhanced resistance of HT cells to these agents, the administration of high concentrations (3.1 µg/ml) of amphotericin B, a distal nephron toxin, killed almost five times more HT cells than isotonic mIMCD-3 cells (Fig. 7E). This effect likely results from the ability of amphotericin B to facilitate sodium entry into cells under conditions of increased extracellular NaCl concentration. Similarly, cadmium chloride, a heavy metal that can induce HSPs, was more toxic to HT cells than to isotonic cells at a dose of 3.13 mM (Fig. 7F). These results indicate that HT cells have acquired a selective stress tolerance or stress-sensitive phenotype characterized by an altered sensitivity to a variety of stresses.


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Fig. 7.   HT cells show selective cross-tolerance of toxic stress. Cytotoxicity was evaluated by crystal violet assay. Selective resistance of HT cells to agents known to induce renal injury including H2O2 (A), cyclosporin (B), mitomycin C (C), amphotericin B (D), cadmium chloride (E), and ceramide (F). HT or isotonic mIMCD-3 cells were exposed to various concentrations of each agent for 24 h. Each bar is the mean ± SE of 14-28 observations. *P < 0.05, Fisher's paired least significant difference (PLSD) test for multiple comparisons shows significance.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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