Chronic hyperosmolarity mediates constitutive expression ofmolecular chaperones - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:33 |只看该作者 |倒序浏览 |打印

作者：Bento C.Santos, James M.Pullman, AlejandroChevaile, William J.Welch,  Steven R.Gullans作者单位：1 Department of Medicine, Brigham and Women‘sHospital, Harvard Institutes of Medicine, Boston 02115; Department of Pathology, University ofMassachusetts Medical School, Worcester, Massachusetts 01655; and Department of Surgery, University of California,San Francisco, California 94143 2 t& P2 h; t3 N' k






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      【摘要】% a: l/ G% b) e. U
   Renal medullary cells areexposed to elevated and variable osmolarities and low oxygen tension.Despite the harsh environment, these cells are resistant to the effectsof many harmful events. To test the hypothesis that this resistance isa consequence of these cells developing a stress tolerance phenotype tosurvive in this milieu, we created osmotically tolerant cells[hypertonic (HT) cells] by gradually adapting murine inner medullarycollecting duct 3 cells to hyperosmotic medium containing NaCl andurea. HT cells have a reduced DNA synthesis rate, with themajority of cells arrested in the G 0 /G 1 phaseof the cell cycle, and show constitutive expression of heat shockprotein 70 that is proportional to the degree of hyperosmolarity.Unlike acute hyperosmolarity, chronic hyperosmolarity failed toactivate MAPKs. Moreover, HT cells acquired protein translationaltolerance to further stress treatment, suggesting that HT cells have anosmotolerant phenotype that is analogous to thermotolerance but is apermanent condition. In addition to osmotic shock, HT cells were moreresistant to heat, H 2 O 2, cyclosporin, andapoptotic inducers, compared with isotonic murine inner medullaryduct 3 cells, but less resistant to amphotericin B and cadmium. HTcells demonstrate that in renal medullary cells, hyperosmotic stressactivates biological processes that confer cross-tolerance to otherstressful conditions.
      【关键词】 heat shock protein nephrotoxins thermotolerance cell cycle inner medullary collecting duct# J. W5 M# \3 e- c
   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 medullaryinterstitium is an important element of this process. As a consequence,cells in the renal medulla must deal with constantly changingextracellular solute concentrations ranging from 300 to 1,200 osM inhumans. In addition to this osmotic stress, the countercurrentsystem results in a reduced oxygen tension in the medulla, confrontingcells 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 thatenables them to adapt to this milieu. This program involves theactivation of signal transduction kinases, synthesis of stressproteins, and accumulation of compatible organic osmolytes, responsiblefor the maintenance of cell volume and intracellular ionic strength( 2, 11, 33 ).
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In addition to withstanding hyperosmolarity, those cells appearresistant to ischemia-reperfusion episodes and manynephrotoxins by mechanisms still unknown ( 14 ).Ischemia-reperfusion injury is a proximal tubule lesion thathas little impact on renal inner medullary cells ( 32 ).Most heavy metals, including cadmium, lead, and mercury, adverselyaffect the proximal tubules but not the collecting ducts( 9 ). These observed differential sensitivities to injuryled us to hypothesize that the physiological adaptation tohyperosmolarity might confer enhanced tolerance of renal medulla cellsto different types of injury, with the induction and constitutive expression of stress-responsive genes.

In contrast to the osmotolerant phenotype of renal medullary cellsobserved in vivo, osmotic stress in vitro has a lethal effect on kidneycells. Exposure of murine inner medullary collecting duct 3 (mIMCD-3)cells, derived from the mIMCD ( 26 ), to hyperosmolar NaClor 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 celldivision ( 28 ). In contrast, mIMCD-3 and Madin-Darby canine kidney cells exposed to an equimolar combination of NaCl and urea aresignificantly 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 tolerancewith a collecting duct cell line (mIMCD-3 cells) adapted tohyperosmolar conditions by a combination of NaCl and urea mimicking theosmotic milieu in the renal medulla. We have termed these hypertonic(HT) cells. In them, biosynthesis rates are slowed and thestress-inducible form of heat shock protein 70 (HSP70) is expressedconstitutively. Furthermore, HT cells exhibit a greater resistance tomany different nephrotoxins. These results with the HT cell linesindicate that the hyperosmotic stress faced by the renal medullaactivates biological mechanisms that result in cells exhibitingcross-tolerance to other stressful conditions.
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MATERIALS AND METHODS6 m4 V; o# M; [

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 every48 h with the addition of 1 ml of a stock solution containingequimolar concentrations of NaCl and urea. At least three differentexperiments with six replicates were performed between cell passages 15 and 52 to obtain the multipleobservations. During this period of time, adapted cells maintained alltheir characteristics, and we have termed these stable lines, at the highest osmolarity (1,270 mosM), HT cells. To assess viability, acrystal violet assay was used. As described before ( 29 ),cells were seeded at 10 4 cells/well in 96-well flat-bottomplates, incubated, and exposed to different treatments. Aftertreatment, DNA of adherent cells was stained with crystal violet. Theviability percentage of treated cells was defined as the absorbancerelative to untreated cells. The percentage of cytotoxicity was definedas 100% (untreated cells) minus the percent viable cells. To confirmviability results, cytotoxicity was also monitored by light microscopicevaluation of both the supernatant and the adherent cells using trypanblue viability assay.
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[ 3 H]Thymidine, [ 3 H]uridine, and[ 3 H]leucine incorporation. Cells were seeded in 96-well plates at 5 × 10 5 cells/well and grown to subconfluence. To measure rates of DNA, RNA, orprotein synthesis, cells received a pulse of labeled substrate (NewEngland Nuclear) as follows: 0.5 µCi/ml [ 3 H]thymidine,1 µCi/ml [ 3 H]uridine, or 0.5 µCi/ml[ 3 H]leucine. After 6 h of exposure to uridine orleucine, or 12 h to thymidine, cells were trypsinized, washed, andharvested by using a 1205 Betaplate System (Wallac, Finland).Incorporation rates were obtained by scintillation counting in thepresence of Betaplate Scint.

Northern blot analysis. As described earlier ( 29 ), cells were washed and total RNAwas 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 theAmerican Type Culture Collection) were labeled with[ 32 P]dCTP using a random hexamer labeling kit(Pharmacia). Blots were prewashed and then hybridized overnight at42°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 consistedof 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serumalbumin), 20 µg/ml salmon sperm DNA, 0.5% SDS, and the 32 P-labeled cDNA probes (10 6 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 HSP70probe detected both inducible (HSP70) and constitutive (heat shockcognate protein 70) transcripts. Autoradiography was performed withReflection TM (NEN Research Products) film and an intensifying screen.1 M8 j3 o) J% @- K* z. o. i: g7 Z! J8 V

Preparation of cell lysates and Western blot analysis. As previously described ( 27 ), cells were washed withice-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 tonitrocellulose (Nitropure, MSD). Membranes were probed with amonoclonal 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 ImagingDensitometer and software).8 E, A5 D+ b3 y. w' y6 b

Casein-affinity chromatography. For chaperone purification, the experiment was performed essentially asdescribed ( 27 ) with slight modifications. A 2-ml column ofcasein conjugated to cyanogen bromide-activated Sepharose (Sigma) wasunfolded by incubation with 6 M urea and 1 M -mercaptoethanol. Theaffinity column was washed and 5 mg of cellular protein (from celllysates) were applied to the column. The following fractions wereobtained and analyzed by SDS-PAGE: 1 ) flow-through,unbounded proteins; 2 ) Mg-wash, wash proteins unspecificallytrapped in the column (last 1.5 ml of the 5-ml wash, an indication ofthe wash effectiveness); 3 ) ATP elution, for putativechaperones elution; and 4 ) acid elution, for proteinsremaining on the column after the ATP elution.5 b* u  @# N* E- E) Q

Preparation of cell lysates and immunoblot analysis to detectintracellular signaling molecules. Cell lysate preparation and immunoblot analysis were performedessentially 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 sodiumfluoride, 10 µg/ml leupeptin, 20 µg/ml antipain, 100 µg/mlbenzamidine, and 10 µg/ml aprotinin. Cell lysates were clarified at15,000 g for 10 min at 4°C, and protein content wasdetermined by using the Bradford microassay (Bio-Rad). Equal amounts ofprotein (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 activeform of ERK1 and ERK2; antiactive JNK polyclonal antibody (1:2,000

romega), which recognizes the phosphorylated active form of the JNKisoforms; and phosphospecific p38 MAPK antibody (1:2,000; New EnglandBiolabs), which recognizes the phosphorylated active form of p38 MAPK." d! C4 X# G/ H9 \5 x% S9 D

Two-dimensional SDS-PAGE. Two-dimensional SDS-PAGE was performed as described previously( 34 ). Briefly, cells were labeled with[ 35 S]methionine (5 µCi) for 12 h in medium lackingmethionine and supplemented with 10% FBS. In addition, cells weresubmitted to heat shock treatment (43°C/1 h) and then returned to37°C and labeled for 12 h with [ 35 S]methionine.Cells were harvested in 0.1% Triton X-100, and equal amounts oflysates were loaded onto pH 5-7 isoelectric-focusing gel. Afterisoelectric focusing, the proteins were resolved by 12.5% SDS-PAGE.Gels were fluorographed and exposed to film.( c6 d; d2 p8 l+ u; _, O: L
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Flow cytometry and cell cycle analysis. Staining was performed according to the Dana Farber Core Flow CytometryCenter protocol. Briefly, cells were cultured in 60-mm plates untilsubconfluence, trypsinized, washed with ice-cold PBS (without divalentcations), and suspended in ice-cold PBS to a concentration of 2 × 10 6 cells/ml. One milliliter of cell suspension wasvortexed while 1 ml of ice-cold 80% ethanol was added in a drop-wisefashion. For fixation, cells were incubated for 30 min on ice. Fixedcells 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 minat 37°C in the dark. Subsequently, the material was submitted to flowcytometric analysis of DNA content and cell cycle progression (ScaliburScan, Becton-Dickinson, and CellFit Software).- Z/ r( ^6 A, }' P

Immunostaining. Cells were grown on sterile glass slides (TechMate Blue Capillary GapMicro Slides, Ventana Medical Systems) in 100-mm petri dishes. Afterconfluence, cells were washed twice with PBS and fixed with ice-coldmethanol 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 mountedon Superfrost Plus slides (Fisher Scientific). The immunostaining wasperformed 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 (AbacusConcepts) by using a t -test and Fisher's pairedleast-significant difference multiple comparisons test whenappropriate. Data are expressed as means ± SE, and significancewas assigned to P 0.05.

RESULTS

Establishment of adapted cells. mIMCD-3 cells were exposed to culture medium containing graduallyincreased amounts of NaCl and urea in equimolar concentrations. Cellswere adapted to a range of osmolarities with the highest osmolarityachieved being 1,270 mosM. These cells survived and retained theirviability at high osmolarity even after storage in liquid nitrogen. Wehave termed these stably adapted lines (1,270 mosM) HT cells.' K5 B5 Z# W; o+ e

Altered biosynthesis and growth properties of HT cells. RNA and protein synthesis rates were measured in cells adapted to arange of osmolarities (Fig. 1 A ). 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. Incontrast, changes in the rate of protein synthesis showed a biphasicbehavior, increasing by up to 65% above control levels when adapted to460 and 610 mosM and decreasing below control levels when mediumosmolarity was 760 mosM or higher. At the highest level of osmolarity(1,270 mosM), the rates of both protein and RNA synthesis were lowest.3 g% E: ~8 [0 N; {' t# |1 t8 A! J
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Fig. 1. Osmotic adaptation gradually affects RNA and proteinsynthesis and leads to decreased cell proliferation and cell cyclearrest. A : rates of RNA (filled bar) and protein (gray bar)synthesis in isotonic (310 mosM) and adapted murine inner medullarycollecting duct 3 (mIMCD-3) cells to hyperosmolarity. Cells were pulselabeled with either [ 3 H]uridine or[ 3 H]leucine for 6 h to determine RNA and proteinsynthesis 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 : comparisonof cell cycle distribution and proliferation rate of hypertonic (HT;1,270 mosM) and isotonic mIMCD-3 cells. Relative numbers of cells indifferent phases of the cell cycle were determined by flow cytometricmeasurements of DNA content (a); each bar represents the mean ± SE of 4 observations. To determine proliferation rates, equal numbersof HT and control mIMCD-3 cells were pulse labeled with[ 3 H]thymidine for 12 h (b); each bar represents themean ± SE of 27 observations (4 different experiments with 7 replicates). * P

Flow cytometric analysis of cellular DNA content (Fig. 1 B,a) showed that HT cells exhibit an increase in the proportion of cellsin G 0 /G 1 phase compared with isotonic cells (68 vs. 57%, P = 0.002) and a corresponding decrease inthe proportion of cells in S (10 vs. 16%, P = 0.006)and G 2 /M phase (23 vs. 28%, P = 0.002). The cell proliferation rate (Fig. 1 B, b), measured as DNAsynthesis, was reduced by 25%, indicating that the altered cell cycleprofile was associated with a reduced rate of cell division. Thusadaptation to hyperosmolarity appears to reduce cell proliferationrates by slowing cell cycle progression at theG 0 /G 1 stage., j/ A  m" R9 y7 w1 e
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Overexpression of HSP70 in HT cells. In renal epithelial cells, acute hyperosmolarity induces the expressionof HSP70 ( 2, 29 ). As shown in Fig. 2, A and B, adaptedcells exhibited a robust expression of inducible HSP70 mRNA (up to 28×increase) and protein. Of note, the HSP70 protein content increased incorrelation with rising medium osmolarity, with highest expressionlevels detectable at highest osmolarity. Constitutive HSP70 (heat shockcognate protein 70) mRNA was increased 1.5 times compared with thecontrol.: l& F  l* B- k. L) B4 o* l

Fig. 2. Heat shock protein 70 (HSP70) expression increased duringthe adaptation of mIMCD-3 cells to osmotic stress without MAPKactivation. A : relative abundance of constitutive (HSC70)and inducible HSP70 mRNA and protein. B : representativeblots showing constitutive HSP70 [heat shock cognate protein 70 (HSC70)] mRNA and inducible HSP70 mRNA and protein expression inisotonic mIMCD-3 cells (310 mosM) and cells adapted to high osmolarity. C : representative blot of proteins eluted from an unfoldedcasein-affinity column probed with anti-HSP70 antibody to determine thechaperone activity of HSP70 in HT cells (1,270 mosM). Protein lysates(Total) from HT cells were applied to the column. Fractions werecollected before (Flow through, unbound fraction) and after sequentialwashing with MgCl 2 (Mg wash), Mg-ATP (ATP elution), andacid (Acid elution). D : representative Western blot analysisof mIMCD-3 cells 30 min after addition of fresh isotonic medium, freshmedium containing 100 mM NaCl, or HT cells. Blots were sequentiallyprobed with an antibody against the phosphorylated forms of ERK, JNK,and p38 MAPK., p9 R6 C! x2 P1 u& R, x  ?! W+ t- W

With the use of a denatured protein-affinity column, the chaperoneactivity of the HSP70 expressed in HT cells was evaluated. In Fig. 2 C, lane 1, the total HSP70 content in proteinlysate from HT cells is shown. Application of this lysate to theunfolded 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 MgCl 2, inthe absence of ATP (Mg wash), very little HSP70 was eluted, indicatingthe specific requirement of ATP for elution. In comparison, addition ofATP to the elution buffer (ATP elution) caused a dramatic release ofHSP70. In the last step (Acid elution), an acid wash failed to eluteany additional HSP70. These data demonstrate that HSP70 constitutivelyexpressed in the HT cells exhibits the functional characteristics of amolecular chaperone.

MAPKs are not activated in HT cells. In renal epithelial cells, acute hyperosmolar stress, elicited byeither urea or NaCl, activates the three principal MAPK cascades,including the ERKs, JNKs, and p38 MAPK ( 33 ). Toevaluate the activation status of MAPK in HT cells, Western blotanalyses were performed by using an antibody against the activephosphorylated forms of ERK, JNK, and p38 MAPK. In isotonic mIMCD-3cells, we did not observe any significant MAPK activity (Fig. 2 D, Isotonic medium), whereas all three MAPKs (ERK, JNK, andp38 MAPK) responded strongly to an acute addition of 100 mM NaCl. Ofnote, in the HT cells no constitutive MAPK activity was observed.However, these kinases became activated when HT cells were acutelyexposed 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.
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Subcellular localization of HSP70. After heat shock treatment, newly synthesized HSP70 protein is known torapidly 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. HSP70immunostaining (Fig. 3 ) revealed littleor no staining in mIMCD-3 cells maintained under isotonic conditions(310 mosM), a result consistent with our Western blot analysis results(Fig. 2 B ). With increasing osmolarity, the cells showed aproportional increase in HSP70 immunostaining (Fig. 3 A ),with the protein being present in both the cytoplasm and the nucleusbut not the nucleolus (Fig. 3 B ). Even under the conditionsof highest osmolarity, no nucleolar staining was observed (Fig. 3 B, 1,270 mosM). In contrast, heat shock treatment ofisotonic mIMCD-3 cells resulted in robust nucleolar immunostaining forHSP70 (Fig. 3 B, Heat). From this last observation, we inferthat osmotic stress, unlike thermal stress, has little or no adverseeffect on the integrity of maturing ribosomes within the nucleolarcompartment.
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Fig. 3. Immunocytochemical localization of inducible HSP70 incells adapted to increasing osmolarities. Osmolarity is indicated foreach panel, except the last, which shows heat shocked cells (Heat). Thepanels labeled 310 mosM show isosmolar cells. Brown3'-3-diaminobenzidine stain shows immunostaining for HSP70, and bluehematoxylin counterstain shows the nuclei. A :low-magnification views showing that HSP70 levels increasedproportionately to the osmolarity of adaptation (magnification, ×500). B : high-magnification views (×5,000) showing that HSP70 waspresent in both nucleus and cytoplasm of adapted cells, but not in thenucleoli, which showed only hematoxylin counterstain. In contrast,transiently heat-shocked (42°C/30 min) isotonic mIMCD-3 cells showedlocalization of HSP70 in nucleoli, as well being present in the nucleusand cytoplasm.
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Localization of HSP70 within the intact kidney. Osmolarity-dependent variation in the expression of HSP70 similar tothat seen in adapted cells was also observed in the intact kidney (Fig. 4 ). HSP70 immunostaining was largelyconfined to the collecting ducts in all regions of the kidney. Theintensity of staining and hence HSP70 levels paralleled thecorticomedullary osmotic gradient and were lowest in the cortex (Fig. 4 A ), intermediate in the outer medulla (Fig. 4 B ),and highest in the inner medulla (Fig. 4 C ). The only otherHSP70 staining observed was in the urothelial lining of the collectingsystem (Fig. 4 C ). Thus osmolarity-dependent constitutiveexpression of HSP70, under isovolemic conditions, is also aproperty of renal medullary cells in vivo.; \( x: m- R1 ^: W

Fig. 4. Immunohistochemical localization of inducible HSP70 insections of rat kidney. Brown 3'-3-diaminobenzidine stain showslocalization of HSP70. A : cortex with minimalimmunostaining. B : outer medulla with increased staining ofcollecting duct. C : inner medulla with strong staining ofcollecting ducts and urothelium of the collecting system.Magnification, ×100.6 u8 S' y8 ^7 T! {: |9 V+ [

Preferential expression of stress proteins in HT cells. After heat shock treatment, preferential translation of mRNAs encodingthe HSPs along with the repression of synthesis of non-stress-relatedproteins has been observed ( 6 ). Because HT cells showedhigh-level expression of inducible HSP70, we examined whether the cellsmight show preferential protein synthesis patterns similar to thatobserved for cells subjected to heat shock treatment. Control mIMCD-3cells and HT cells were labeled to steady state with[ 35 S]methionine. Cell lysates were prepared, and thepattern of protein synthesis was examined by two-dimensionalelectrophoresis. As seen in Fig. 5, theamount and the pattern of proteins being synthesized were indeeddifferent in the HT cells (Fig. 5 B ) compared with mIMCD-3cells (Fig. 5 A ). Although the gels were loaded with the sameamount of total protein, the overall content of radiolabeled proteinsappeared higher in the mIMCD-3 cells compared with the HT cells. Thisobservation is consistent with those presented in Fig. 1 A showing that HT cells displayed a dramatically reduced rate of overallprotein synthesis. Note as well that the general pattern of proteinsynthesis was significantly different between the mIMCD-3 cells and theHT cells. Specifically, the HT cells showed high-level expression of aselect group of proteins, including HSP70, along with a reducedexpression of other proteins that were expressed in the mIMCD-3 cells.
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Fig. 5. Two-dimensional gel electrophoresis of 35 S-labeledproteins isolated from control mIMCD-3 cells or HT cells, with orwithout heat shock treatment. Control mIMCD-3 cells ( A ) orHT cells ( B ) were pulse labeled with[ 35 S]methionine for 12 h. In addition, mIMCD-3 cells( C ) or HT cells ( D ) were submitted to heat shocktreatment (43°C/1 h) and then returned to 37°C and labeled for12 h with [ 35 S]methionine. Gels were loaded withidentical amounts of protein. Downward arrow, HSC70; upward arrow,HSP70.
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Two-dimensional gel analysis of mIMCD-3 cells after a 43°C/1 h heatshock treatment revealed the expected increase in inducible HSP70expression along with the reduced expression of nonstress-related proteins in the heat shock-treated mIMCD-3 cells compared with theirnonheated counterparts (Fig. 5, C vs. A,respectively). In contrast, heat shock treatment of the HT cells didnot result in any major changes in the protein expression. No furtherincrease in HSP70 expression was observed nor were there anysignificant effects on the expression of other proteins (Fig. 5, D and B ). These observations areconsistent with the idea that HT cells have adopted a stress-likephenotype characteristic of cells subjected to transient hyperthermia.
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HT cells exhibit enhanced stress tolerance. In all organisms, exposure to a mild heat shock treatment elicits atransient state of thermoresistance known as "acquired thermotolerance." Moreover, cells can exhibit "cross-tolerance"; transient exposure to one stressor can confer enhanced tolerance toothers ( 23 ). The degree of acquired tolerance correlates with the expression levels of different HSPs, particularly HSP70 ( 13 ). Therefore, we examined whether HT cells mightexhibit 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. 6 A ). Assessment ofcytotoxicity 24 h after treatment clearly demonstrated that HTcells were less heat sensitive than isotonic cells (15 vs. 56%, P addition, we analyzed whether HT cellscould tolerate acute hyperosmotic stress, a circumstance similar tothat occurring in vivo during transitions in hydration state. As shownin Fig. 6, B - D, HT cells were more resistantto acute addition of NaCl, urea, or a combination of NaCl and ureacompared with isotonic cells. Furthermore, the maximal osmolarities(i.e., combination of NaCl and urea) tolerated by at least 50% of thecells for 24 h were 2,540 mosM for HT cells but only 750 mosM forthe isotonic cells.

Fig. 6. HTcells are more tolerant of osmotic and thermal stress. Viability of HTand control mIMCD-3 cells after acute heat stress or hyperosmoticstress was evaluated by crystal violet assay. A : mIMCD-3cells or HT cells were exposed to severe heat shock (46°C/4 h); barsrepresent the mean value ± SE of 50 and 89 observations,respectively. The differences in sensitivity observed between isotonicand HT cells were statistically significant ( P B ), urea ( C ), or NaCl urea ( D ). Eachpoint is the mean ± SE of 17 observations. Statisticalsignificance of differences was tested by using Fisher's paired leastsignificant difference test for multiple comparisons(* P! R: X. `- F  X  m* E6 f& A* R! X$ p
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We tested the resistance of HT cells to nephrotoxins (Fig. 7 ), including therapeutic drugs, oxidantstress, and heavy metal compounds. Interestingly, HT cells exhibited avery diverse sensitivity to these different noxious agents. Hydrogenperoxide (Fig. 7 A ), used to mimicischemia-reperfusion injury, was considerably less cytotoxic tothe HT cells than to isotonic mIMCD-3 cells. Similar stress resistancewas observed after exposure to cyclosporin (Fig. 7 B ), withnearly 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 thetreatment of solid tumors, which can cause glomerular and proximaltubular but not inner medullary injuries in vivo ( 3 ) (Fig. 7 C ), was less cytotoxic to HT cells than to isotonic controlcells. Ceramide (Fig. 7 D ), an inducer of apoptosisimplicated 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 and100 µM). In contrast to the enhanced resistance of HT cells to theseagents, the administration of high concentrations (3.1 µg/ml) ofamphotericin B, a distal nephron toxin, killed almost five times moreHT cells than isotonic mIMCD-3 cells (Fig. 7 E ). This effectlikely results from the ability of amphotericin B to facilitate sodiumentry into cells under conditions of increased extracellular NaClconcentration. Similarly, cadmium chloride, a heavy metal that caninduce HSPs, was more toxic to HT cells than to isotonic cells at adose of 3.13 mM (Fig. 7 F ). These results indicate that HTcells have acquired a selective stress tolerance or stress-sensitivephenotype characterized by an altered sensitivity to a variety ofstresses.
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Fig. 7. HT cells show selective cross-tolerance of toxic stress.Cytotoxicity was evaluated by crystal violet assay. Selectiveresistance of HT cells to agents known to induce renal injury includingH 2 O 2 ( A ), cyclosporin( B ), mitomycin C ( C ), amphotericin B( D ), cadmium chloride ( E ), and ceramide( F ). HT or isotonic mIMCD-3 cells were exposed to variousconcentrations of each agent for 24 h. Each bar is the mean ± SE of 14-28 observations. * P
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DISCUSSION5 U4 j1 L! E5 `
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Medullary hyperosmolarity and hypoxia are an inevitableaccompaniment 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, bygradually adapting mIMCD-3 cells to a hyperosmotic environment up to1,270 mosM in vitro. In contrast to the apoptotic effect of highextracellular concentrations of either NaCl or urea, a combination ofboth, as exists in vivo, promoted such an acclimation process( 21, 28, 39 ).

In HT cells, DNA synthesis was reduced by 25%, suggesting a decreasedrate of cell division (Fig. 1 B, b). Consistent with this, HTcells exhibited an increase in the proportion of cells within theG 0 /G 1 stage and a corresponding decrease incells in the S and G 2 /M phases (Fig. 1 B, a). Ithas been shown that during the G 1 phase, cells respond toextracellular signals by either advancing toward another division orwithdrawing from the cycle into a resting state (G 0 )( 31 ). Thus adaptation to hyperosmolarity appears to slowproliferation by delaying the exit from theG 0 /G 1 stage. This response may be mediated, inpart, through changes in p53, as described in acute NaCl stress,determining cell growth arrest and protection from apoptosis( 4 ).
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With increasing extracellular osmolarity, the rate of RNA synthesisdecreased (Fig. 1 A ). In contrast, the rate of protein synthesis showed a biphasic profile depending on the cell culture medium osmolarity (Fig. 1 A ). In cells adapted to lowerosmotic conditions, protein synthesis was increased, whereas underhigher 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.0 u2 i6 X: x  C9 Z" M1 i! ~* C

Increased expression of HSPs and molecular chaperones is a ubiquitousfeature of cells exposed to acute, but typically transient, stressconditions. In contrast, a constitutive high level of inducible HSPs(e.g., HSP70) is not well known, particularly in mammals. In a numberof organisms, induced expression of HSPs can be a marker for theadaptation to cyclic environmental changes, as well as a mechanism thatallows different species to live in especially harsh environments( 8 ). We found that a progressive increase in theexpression of HSP70 paralleled the adaptation of the cells to highosmotic stress (Figs. 2 and 3 ). A comparable relationship was alsofound in vivo, where levels of inducible HSP70 expression followed thecorticomedullary osmotic gradient (Fig. 4 ).; R& j! Z  R1 N, ]5 T+ N7 P: u
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The use of heterologous promoters to force HSP70 expression has shownthat constitutively expressed HSP70 coalesces into granules, and theprotein present in these granules appeared to be irreversibly inactivated. It could not be dispersed with a second heat shock, andcells containing these granules did not show thermotolerance ( 7 ). Performing experiments with a denaturedprotein-affinity column, we showed that HSP70 that accumulates in HTcells possesses chaperone activity. Subcellular localization of HSP70in cells chronically exposed to osmotic stress was comparable to thatobserved 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, whichappeared to be independent of the severity of the osmotic stress, mostlikely reflects similar subcellular roles for HSP70 in acutely andchronically stressed cells (Fig. 3 B ). In contrast toheat-shocked mIMCD-3 cells (Fig. 3 B, Heat), the absence ofnucleolar localization of HSP70 in HT cells suggests that there is nochange in nucleolar activity in osmotic stress. Nevertheless, the highlevels of HSP70 seen in HT cells most likely represent the reason forthe increased tolerance of these cells to hyperosmotic (Figs. 6, B - D ) as well as heat (Fig. 6 A )stress. This hypothesis is strengthened by the observation that thedegree of osmotic tolerance is proportional to the level of osmolaradaptation and consequently to the cellular content of HSP70 protein(data not shown).* U4 |/ p7 Q# O  }5 Y& ?
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In contrast to the hypertonic activation of MAPKs observed after acutetreatment ( 33 ), no active MAPKs were detectable in HTcells. This, together with the observation that other acute stresseswere able to elicit MAPK activity in HT cells (data not shown),suggests that MAPKs are primarily involved in the acute response tohyperosmotic stress. This in vitro observation implies that theconstitutive activation of MAPKs detected in vivo in inner medullarycells ( 36 ) may be secondary to other stress factors foundin the inner medulla and not related to the chronic environmentalhyperosmolarity in this kidney zone. Although MAPK activation has beenlinked to HSP expression in response to acute hyperosmolarity( 30 ), our data suggest that MAPKs are not crucial in theregulation of the constitutive expression of HSP70 induced by chronichyperosmolar stress.

As a typical response to a severe heat shock, normal cellulartranslational activity is repressed. After heat shock, cells recoverfull 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 eventsare key determinants of the phenomenon of thermotolerance. Evaluationof the protein expression pattern in HT cells disclosed an overalldecrease in protein synthesis. Interestingly, a superimposed heat shockdid 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 recoveryof general protein synthesis starts as soon as a certain threshold ofHSP70 expression is attained ( 6 ). Therefore, the translational tolerance observed in HT cells might be related to thehigh levels of HSP70 expression.' _) ~( n0 j" R: \2 l

For analysis of the stress-tolerant phenotype, we tested the resistanceof HT cells to known nephrotoxins, including therapeutic drugs, oxidantstress, and heavy metal compounds. Those stressors have in common thecapacity of inducing cell death by apoptosis ( 12, 16-18, 24, 25, 28 ). In addition, there is evidence thatHSPs play a role in apoptosis, because overexpression of HSP70can prevent apoptosis ( 10, 20 ). We showed that HTcells are more resistant to toxic levels of osmotic, heat, oxidative (H 2 O 2 ), cyclosporin, and mitomycin C stressthan isotonic mIMCD-3 cells, suggesting the development of across-tolerant phenotype to many other stressors. On the other hand,high doses of ceramide (a sphingolipid metabolite and an inducer ofapoptosis), amphotericin B, or cadmium were more toxic for HTcells than isotonic cells. The cell injury induced by amphotericin B isdetermined by cell membrane modification and augmented inorganic ioninflux ( 18 ), and the increased sensitivity of HT cells toamphotericin B in vitro correlates with the known toxicity ofamphotericin B for IMCD cells in vivo ( 5 ), suggesting thatthis substance is only toxic to IMCD cells in combination with osmoticstress. Of note, cadmium is not toxic to IMCD cells in vivo, but thisappears to be due to the induction of metallothionase enzymes in theproximal tubules, which are known to chelate and detoxify cadmiumbefore it reaches the IMCDs ( 15 ). Heat shock is known tocross protect against heavy metals, yet we did not find this to be thecase in HT cells. This discrepancy might be explained by differences wehave 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 nodirect correlation between HSP70 levels and cell survival.
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In conclusion, the present investigation demonstrates that aphysiological condition, namely, hyperosmolarity, can elicit a stresstolerant phenotype in mammalian cells. Using HT cells as a model, thiscellular phenotype is characterized by reduced biosynthetic andcell-division rates, related to a slowed cell cycle progression,constitutively high levels of inducible HSP70, and translationalthermotolerance, in the absence of overt activation of MAPK signaling.The antiproliferative effect, observed in association with thehyperosmolar adaptation of HT cells, as well as the high levels ofHSP70, most likely represents mechanisms enabling cells to survive inthe harsh renal medullary environment. Because HT cells werecross-tolerant of several stress factors other than hyperosmoticstress, one can conclude that the osmotolerant phenotype may contributeto the ability of renal medullary cells to also withstand injuries thatare detrimental to other regions of the kidney and other organs.Therefore, further studies of this adaptive mechanism might open newapproaches in the prevention or treatment of acute renal injury.' ?, y. d1 T* z) p+ f/ F! F  I
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ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes andDigestive 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).; O4 f* b4 N& T; v
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