Regulatory volume increase is associated with p38 kinase-dependent actin cytoske - 《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 13:40 |只看该作者 |倒序浏览 |打印

作者：Mauro Bustamante, Frank Roger, Marie-Luce Bochaton-Piallat, Giulio Gabbiani, Pierre-Yves Martin,  Eric Féraille作者单位：1 Division de Néphrologie, Fondation pourRecherches Médicales, and Départementde Pathologie, Centre Médical Universitaire, CH-1211 Genève Switzerland & p  @/ M5 x' I$ C3 i

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      【摘要】
   The kidney medulla is physiologically exposed to variations inextracellular osmolality. In response to hypertonic cell shrinkage, cells ofthe rat kidney medullary thick ascending limb of Henle's loop undergo p38kinase-dependent regulatory volume increase (RVI). In the present study, weinvestigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases andreduces cellular volume, induced a sustained actin polymerization occurringafter 10 min and concurrently with RVI. In contrast, hyperosmotic urea, whichdoes not modify MAP kinase activity and cellular volume, did not inducesustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution ofF-actin from a dense cortical ring to a diffuse network of actin bundles.Stabilization of actin filaments by jasplakinolide and inhibition of thegeneration of new actin filaments by swinholide A prevented RVI, whereasdepolymerization of actin filaments by latrunculin B attenuated cell shrinkageand enhanced RVI. These actin-interfering drugs did not alter extracellular regulated kinase and p38 kinase activation under hypertonic conditions.Similar to swinholide A, inhibiting p38 kinase with SB-203580 abolishedsustained actin polymerization, actin redistribution, and decreased RVIefficacy. We therefore propose that in rat kidney the medullary thickascending limb of Henle's loop exposed to extracellular hypertonicity, p38kinase activation induces depolymerization of the F-actin cortical ring andpolymerization of a dense diffuse F-actin network that both contribute to increase RVI efficacy.
      【关键词】 mitogenactivated protein kinase osmolarity kidney medulla cell volume
   DURING DIURESIS AND antidiuresis, the kidney medulla ofvertebrates is exposed to large fluctuations in interstitial osmolality ( 23 ), which challenges cellvolume constancy. Under antidiuresis, the countercurrent concentrationmechanism initiated by active NaCl reabsorption by the medullary thickascending limb of Henle's loop (MTAL) leads to NaCl and urea accumulation in the renal medulla. MTAL cells are therefore of special interest because theyhave developed adaptive mechanisms to survive and function in a hypertonicenvironment. The high interstitial osmolality induces changes in the activityof solute transporters and enzymes involved in solute accumulation and in theexpression of genes encoding enzymes required for solute synthesis, stress resistance, and yeast cell wall structure( 34 ). After cell shrinkage inresponse to extracellular hypertonicity, MTAL cells progressively recovertheir initial volume through regulatory volume increase (RVI). This processoccurs within minutes and is mediated by stimulation of ion transporters thatincrease intracellular ionic concentrations, which drive water influx andrestore initial cellular volume( 34, 43 ). The rapid increase inionic concentrations is followed by a slow accumulation of intracellularcompatible osmolytes, such as sorbitol, myoinositol, taurine, betaine, andglycerophosphocholine, allowing recovery of normal ionic concentrations( 7 ). This process is along-term mechanism occurring within hours or days and counteracts increased extracellular osmotic pressure.
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Hypertonic conditions activate MAPK, which is an important signal transducer linking signals from the cell surface to the nucleus. MAPKs areserine/threonine kinases activated by a cascade of kinases involving twoupstream kinases, MAPKKK and MAPKK( 48 ). In mammalian cells,MAPKs are divided into three families, each responding to distinctextracellular stimuli: ERK 1 and 2, JNK (also known as stress-activatedprotein kinases 1), and p38 kinases (or stress-activated protein kinase 2). In our previous study, we showed that cell shrinkage, rather than intracellularhypertonicity, triggers the activation of ERK and p38 kinase in rat MTALs( 35 ). MAPK activation levelswere dependent on the osmolyte used to increase extracellular osmolality. Hyperosmotic NaCl induced cell shrinkage and activated ERK and p38 kinase butnot JNK. In comparison, hyperosmotic sucrose induced even greater cellshrinkage and stronger activation of ERK and p38 kinase and also activated JNKbut to a lesser extent. By contrast, hyperosmotic urea altered neither cell volume nor MAPK activity. Both hypertonic NaCl and sucrose triggered cellularRVI that restored, almost completely for NaCl and partially for sucrose, theinitial cellular volume. Inhibition of p38 kinase decreased the efficiency ofRVI, implying a major role of this kinase in this process, whereas inhibitionof ERK did not alter RVI.
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Modifications of cellular architecture related to hypertonicity-induced cell shrinkage are associated with a reorganization of the architecture of theactin cytoskeleton and with changes in the F-actin-G-actin equilibrium( 13, 18, 19, 32, 39 ). Specific cytoskeleton components may sense cell volume decrease and initiate signaling cascadesleading to RVI. In addition, signal transduction cascades leading toremodeling of the actin cytoskeleton and to MAPK activation share some commonelements. For instance, small G proteins of the Rho family, such as Cdc42 andRac 1, are involved in both actin cytoskeleton remodeling through filipodia and lamellipodia formation( 45 ) and in signaling eventsleading to p38 kinase activation( 1, 51 ). Activation of p38 kinase may, in turn, control actin cytoskeleton dynamics through the activation ofdownstream kinases such as MAPKAP kinase 2/3 or PRAK, which phosphorylate HSP25/27 ( 17, 28, 37 ), a small heat shockprotein that modulates actin polymerization( 27 ). The actin cytoskeletonmay also control the activity of ion transporters, leading to intracellularNaCl uptake and secondary water influx, either directly, throughF-actin/G-actin ratio dynamics( 9, 10 ), or indirectly, throughbinding of signaling modules( 50 ) and/or modulation ofendocytotic-exocytotic events ( 42 ). This study was thereforeundertaken to investigate the relationship between actin cytoskeletonremodeling and RVI in rat MTAL.

MATERIALS AND METHODS
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Preparation of single MTALs. Male Wistar rats weighing150-200 g were anesthetized with pentobarbital sodium (5 mg/100 g body wt ip), and the left kidney was immediately removed after perfusion withice-cold incubation solution (120 mM NaCl, 5 mM RbCl, 4 mM NaHCO 3,1 mM CaCl 2, 1 mM MgSO 4, 0.2 mMNaH 2 PO 4, 0.15 mM Na 2 HPO 4, 5 mMglucose, 10 mM lactate, 1 mM pyruvate, 4 mM essential and nonessential aminoacids, 0.03 mM vitamin, 20 mM HEPES, and 0.1% BSA, pH 7.4) containing 0.18%(wt/vol) collagenase. After incubation at 30°C for 20 min in incubationsolution (see above) containing 0.05% (wt/vol) collagenase, kidney slices werestored at 4°C, and single MTALs were microdissected understereomicroscopic control in oxygenated (95% O 2 -5% CO 2 ) incubation solution.
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Preparation of MTAL suspensions. The two kidneys were perfused with ice-cold incubation solution without collagenase. The inner stripes ofthe outer medulla were excised, minced on ice, and fragments of medullarytubules were obtained by gentle pressure through nylon filters with a poresize decreasing from 150 to 100 µm. After centrifugation, the pellet wasresuspended in ice-cold oxygenated (95% O 2 -5% CO 2 )incubation solution. As controlled under a stereomicroscope, MTALs account for 90% of the tubule fragments in this preparation. Therefore, it will bereferred as MTAL suspension.

Determination of the Triton X-100-soluble and -insoluble actin fractions. Estimation of actin polymerization level was performed bydetermining the Triton X (TX)-100-soluble/TX-100-insoluble actin ratio.Indeed, it is largely admitted that F-actin, i.e., polymerized actin, iscontained in the TX-100-insoluble fraction and that G-actin, i.e., monomericactin, is contained in the TX-100-soluble fraction( 16 ). After 1-h preincubationat 30°C in isotonic incubation solution with or without addition of drugs,MTAL suspensions were incubated at 37°C for 1 to 30 min under isosmotic orhyperosmotic (addition of 300 mosM/l NaCl, sucrose, or urea) conditions.Incubation was stopped by cooling and centrifugation at 6,000 g for 5min at 4°C. The pellet was saved and 20 µl of ice-cold lysis buffer (20mM Tris · HCl, 2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mMNa 4 O 7 P 2, 2 mM Na 3 VO 4, 1mM AEBSF, 10 µg/ml leupeptin, 4 µg/ml aprotinin, 1% Triton X-100, pH7.45) were added. After 5 min of centrifugation at 12,000 g, thesupernatant was saved, the pellet was then mixed with fresh lysis buffer, andafter a centrifugation step at 12,000 g, the second supernatant wassaved. The final pellet was then suspended in sample buffer and an equalvolume of sample buffer was added to the pooled supernatants. The proteinsfrom pooled supernatants and pellet were then separated by 10% SDS-PAGE andtransferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P,Millipore, Waters, MA), and -actin was detected by immunoblot using amonoclonal anti- -actin antibody (AC-15, Sigma, St. Louis, MO) at1:40,000 dilution (vol/vol). After incubation with anti-mouse IgG coupled tohorseradish peroxidase (Transduction Laboratories, Lexington, UK) at 1:10,000dilution (vol/vol), immunoreactivity was detected by chemiluminescence usingthe Super Signal Substrate method (Pierce, Rockford, IL). Results werequantified under conditions of linearity by integration of the density oftotal area of each band using a video densitometer and Image-Quant software (Molecular Dynamics, Sunnyvale, CA). Results are expressed as a percentage ofthe control optical density (isotonic medium) and are means ± SE.5 }  p; w4 g$ l6 u! E2 ]+ R7 c

Determination of the phosphorylation level of ERK and p38 kinase. After 1-h preincubation at 30°C in isotonic incubation solution with orwithout addition of drugs, MTAL suspensions were incubated at 37°C for 10min under isosmotic or hyperosmotic (addition of 300 mosM/l NaCl) conditions.Incubation was stopped by cooling and centrifugation at 6,000 g for 5min at 4°C. After addition of lysis buffer, protein content was measuredby the BCA protein assay (Pierce). Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Immobilion-P,Millipore). Phosphorylated ERK and p38 kinase were detected using anti-ERK-Pand anti-p38-P kinase rabbit polyclonal antibodies (New England Biolabs,Beverly, MA) at 1:10,000 dilution (vol/vol). After incubation with anti-rabbit IgG coupled to horseradish peroxidase (Transduction Laboratories) at 1:10,000dilution (vol/vol), immunoreactivity was detected by chemiluminescence, andresults were quantified and expressed as described above.
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Determination of MTAL cellular volume. A pool of three isolated MTALs was transferred into the concavity of a bacteriological slide coatedwith dried BSA. After 1-h preincubation at 30°C in isosmotic incubationsolution with or without addition of drugs, MTALs were incubated at 37°Cfor 1 to 30 min under isosmotic or hyperosmotic (addition of 300 mosM/l NaCl)conditions. After preincubation at 30°C in isosmotic incubation solution, tubules were incubated in isosmotic or hyperosmotic incubation solutions withor without drugs. MTALs were visualized with an inverted microscope, andphotographs of the same tubules were taken at the end of the preincubationperiod and after incubation. MTAL volume (V) was calculated from the measured radius ( R ) and length ( L ) of the tubules at a 1,000-foldmagnification using the formula V = R 2 x L. Because the lumen is collapsed in nonperfused tubules, we assumedthat MTAL volume measurement is an appropriate estimate of MTAL cellularvolume. Results are expressed as a percentage of the control volume (end of the preincubation period) and are means ± SE.% x: c. H1 R$ {" p( J

Fluorescence microscopy. MTAL suspensions were preincubated at30°C for 1 h with or without drugs and then incubated at 37°C underisosmotic or hyperosmotic (addition of 300 mosM/l NaCl or sucrose) conditions.Tubules were then cytocentrifuged on glass slides using a cytospin (70 g, 5 min in incubation solution supplemented with 1% BSA) and fixedwith 3.7% paraformaldehyde for 10 min at room temperature. After three washesin PBS, fixed tubules were permeabilized with 0.1% Triton X-100 for 1 min atroom temperature. After a new series of three washes in PBS, specimens wereincubated with phalloidin Alexa-488 (dilution: 1:100 in PBS; Molecular Probes,Eugene, OR) for 1 h at room temperature. Specimens were observed with a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany) equipped with an oil-immersionplan-neofluar x 40:1.3 objective. Images were acquired with ahigh-sensitivity, high-resolution color camera (Axiocam, Carl Zeiss). Pictureswere printed with a digital pictrography 4000 printer (Fujifilm, Tokyo,Japan).
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Statistical analysis. Statistical analysis of variations of TX-100-insoluble/TX-100-soluble actin and cellular volume was done by ANOVA.Statistical analysis of variations of anti-P-ERK and P-p38 kinaseimmunoreactivity was done using the Kruskall-Wallis test. P values

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Actin cytoskeleton remodeling in response to hyperosmotic NaCl, sucrose, or urea. We previously showed that hyperosmotic NaCl andsucrose, but not urea, induced both MAPK activation and cell shrinkage in ratMTAL cells ( 35 ). Because cellshrinkage and/or intracellular hypertonicity may induce remodeling of theactin cytoskeleton, we compared the time course of the effects of hyperosmoticNaCl, sucrose, and urea on the F-actin/G-actin ratio and cellular volume inrat MTALs.
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Figure 1 shows thatincreasing extracellular osmolarity up to 600 mosM/l by addition of NaCl,sucrose, or urea rapidly increased the proportion of TX-100-insoluble actin,i.e., F-actin, with a peak observed after 1- to 3-min incubation at 37°C. The TX-100-insoluble/TX-100-soluble actin ratio then returned close to itsbasal level after 10-min incubation. These rapid variations in cellularTX-100-insoluble actin content were followed by a progressive increase inproportion of TX-100-insoluble actin above the basal levels in samplesincubated up to 30 min in the presence of hyperosmotic NaCl(TX-100-insoluble/TX-100-soluble actin; isosmotic: 1.46 ± 0.18; NaCl:2.46 ± 0.30; P actin was sustained for at least 60 min (data not shown). The progressive increase in proportion of TX-100-insoluble actin was morepronounced after 30 min in the presence of hyperosmotic sucrose(TX-100-insoluble/TX-100-soluble actin; isotonic: 2.10 ± 0.37; sucrose:5.98 ± 1.03; P with hyperosmotic NaCl( Fig. 1, A and B ). In contrast, for incubation periods ranging from 10to 30 min, hyperosmotic urea did not induce significant variations in theTX-100-insoluble/TX-100-soluble actin ratio (isotonic 30 min: 1.75 ±0.11; urea 30 min: 1.60 ± 0.12; not significant; Fig. 1 C ). As shownpreviously ( 35 ), bothhyperosmotic NaCl and sucrose rapidly induced cell shrinkage with a maximaldecrease in cellular volume observed after 10 min of incubation (% of initialcellular volume; NaCl: 68.98 ± 2.21; sucrose: 65.47 ± 0.58).After 30-min incubation in the presence of hyperosmotic NaCl or sucrose, apartial recovery of the initial cellular volume was observed (NaCl: 89.14± 2.24; sucrose: 81.81 ± 5.94; Fig. 1, A and B ). In contrast, hyperosmotic urea did not significantly alter cellular volume ( Fig.1 C ). These results show that, in MTAL cells, acuteextracellular hyperosmolality induces polyphasic actin cytoskeleton remodelingreflected by the observed changes in the F-actin/G-actin ratio. However,sustained actin polymerization reflected by the progressive increase incellular F-actin content was only observed in response to osmolytes inducingcell shrinkage and this event occurred concomitantly with the partial recovery of the initial cellular volume.# T: e' g  i# h$ Z
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Fig. 1. Effect of hyperosmotic NaCl, sucrose, and urea on actin polymerizationlevel and cellular volume. Medullary thick ascending limb of Henle's loop(MTAL) suspensions or microdissected MTALs were incubated under isosmotic( time 0 )or hyperosmotic (600 mosM/l for 1 to 30 min) conditions. -Actin sorting between Triton (TX)-100-insoluble (i) and -soluble (s)fractions was analyzed by Western blot analysis and theTX-100-insoluble/soluble actin ratio was calculated after quantification bydensitometry. Cellular volume was determined from photographs. Results aremeans ± SE from 6 to 11 independent experiments (* P time 0 ). The graphs show the time course ofTX-100-insoluble/soluble actin ratio ( left axis) and cellular volume( right axis) variations after addition of hyperosmotic NaCl( A ), hyperosmotic sucrose ( B ), or hyperosmotic urea( C ). Insets : representative Western blot analysesillustrating actin sorting after 30-min incubation in the presence ofhyperosmotic NaCl, hyperosmotic sucrose, or hyperosmotic urea.5 u) U# B. P$ H) m6 Q6 f5 f5 W
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The effect of extracellular hyperosmolality on actin cytoskeleton organization was assessed by fluorescence microscopy on isolated rat MTALsincubated at 37°C for 30 min under isosmotic or hyperosmotic conditions.As shown by Fig. 2 A,rat MTAL cells incubated under isosmotic conditions exhibited a dense corticalF-actin ring delineating the cell periphery and a sparse diffuse network ofF-actin bundles. After exposure of MTALs to hyperosmotic NaCl, the corticalF-actin ring was thinner and the diffuse F-actin network was more developedcompared with tubules incubated under isosmotic conditions( Fig. 2 B ). Thisredistribution of F-actin was more pronounced after incubation of tubules inthe presence of hyperosmotic sucrose ( Fig.2 C ). In contrast, hyperosmotic urea did not induce anysignificant change in F-actin distribution( Fig. 2 D ). Therefore,the sustained actin polymerization phase observed in response to hyperosmotic NaCl and sucrose was associated with a redistribution of F-actin from thecortical F-actin ring to a diffuse network of F-actin bundles. In contrast,the early actin polymerization phase was not associated with apparent F-actinredistribution (data not shown).
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Fig. 2. Effect of hyperosmotic NaCl, sucrose, and urea on actin cytoskeletonorganization. MTAL suspensions were incubated for 30 min under isosmotic( A ) or hyperosmotic conditions (600 mosM/l) with addition of NaCl( B ), sucrose ( C ), or urea ( D ). Tubules were thenfixed with 4% paraformaldehyde, permeabilized by 0.1% TX-100, and Factin wasvisualized by fluorescence microscopy after incubation with phalloidin-Alexa488. Representative en face views of MTAL epithelium are shown. A :under isosmotic conditions, MTAL cells exhibit a dense cortical F-actin ring(filled arrow) and a sparse diffuse network of F-actin bundles (dashed arrow).Both hyperosmotic NaCl ( B ) and hyperosmotic sucrose ( C )induced redistribution of F-actin from the cortical ring to a dense diffusenetwork of F-actin bundles, whereas hyperosmotic urea ( D ) did notalter actin cytoskeleton morphology.! ~, X9 F+ |. O, ]5 [, [$ l/ |
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Interfering with actin polymerization and remodeling altered cellularvolume recovery under hypertonic conditions. The actin cytoskeleton is ahighly dynamic structure that undergoes constant remodeling, consisting ofspatially and temporally regulated polymerization and depolymerization ofpreexisting filaments as well as nucleation and branching of new actinfilaments. New pharmacological tools derived from marine sponges( 40 ) were used to study therole of actin polymerization-depolymerization and generation of new actinfilaments on cellular volume recovery under hypertonic conditions.Jasplakinolide binds to both ends of actin filaments preventing theirdepolymerization and also causes rapid nucleation of actin polymerization( 5, 40 ). As expected from itsmechanism of action, 10 µM jasplakinolide (Calbiochem, San Diego, CA)increased the proportion of TX-100-insoluble actin in tubules incubated underhypertonic conditions for 30 min (TX-100-insoluble/TX-100-soluble actin; NaCl:2.87 ± 0.49; NaCl jasplakinolide: 4.02 ± 0.45; P Fig. 3 A ). Inaddition, fluorescence microscopy revealed that jasplakinolide-treated tubulesexhibited a very dense F-actin network with diffuse thick and short actin-richstructures, i.e., actin clumps, throughout the cytoplasm( Fig. 3 B ). Therefore, jasplakinolide efficiently increases the actin polymerization in rat MTALcells. Swinholide A inhibits actin filament nucleation and elongation( 6, 40 ), thereby preventingstimulus-induced actin polymerization without affecting the intact actinnetwork ( 46 ). Measurement ofthe partition of actin between TX-100-soluble and -insoluble fractions showedthat, in agreement with its pharmacological properties, 50 µM swinholide A(Calbiochem) moderately decreased the amounts of TX-100-insoluble actin measured after 30-min incubation under hypertonic conditions (TX-100-insoluble/TX-100-soluble actin; NaCl: 2.87 ± 0.49; NaCl  swinholide: 2.17 ± 0.28; not significant; Fig. 3 A ). Fluorescencemicroscopy, however, showed that reorganization of the actin cytoskeletoninduced by hyperosmotic NaCl was largely prevented by swinholide A (compare a and c, Fig.3 B ). Indeed, most F-actin remained in the cortical ringand the density of the diffuse F-actin network was unchanged compared withMTALs incubated under isotonic conditions. Therefore, swinholide A does notsignificantly alter actin cytoskeleton organization but prevents itshypertonicity-induced remodeling in rat MTAL cells. Latrunculin B sequestersmonomeric actin and decreases G-actin availability, resulting in actinfilament depolymerization ( 40, 41 ). Consistent with itsactin-depolymerizing properties, 100 µg/ml latrunculin B (Calbiochem)induced a large decrease in proportion to TX-100-insoluble actin with respectto control after 30-min incubation under hypertonic conditions(TX-100-insoluble/TX-100-soluble actin; NaCl: 2.87 ± 0.49; NaCl  latrunculin: 0.33 ± 0.09; P 0.01; Fig. 3 A ). In addition,fluorescence microscopy revealed that latrunculin B disorganized the actincytoskeleton. The cortical F-actin ring became irregular and discontinuous,and the diffuse F-actin network was almost completely disrupted (compare a and d, Fig.3 B ). Therefore, latrunculin B potently depolymerizes theactin cytoskeleton in rat MTAL cells.$ Y7 E7 D! o& g8 q. f
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Fig. 3. Effect of jasplakinolide, swinholide A, and latrunculin B on actinpolymerization level and actin cytoskeleton reorganization in response toextracellular hypertonicity. MTAL suspensions were incubated underhyperosmotic (600 mM NaCl) conditions without (control; H) or with 10 µMjasplakinolide, 50 µM swinholide A, or 100 µg/ml latrunculin B. A : actin sorting between TX-100-insoluble and -soluble fractions. TheTX-100-insoluble/soluble actin ratio was determined as described in the legendof Fig. 1 and results are means± SE from 3 independent experiments (* P -actin sorting in theabsence or presence of drugs is shown. B : fluorescence imaging of theactin cytoskeleton. After 30-min incubation, tubules were fixed with 4%paraformaldehyde, permeabilized by 0.1% TX-100, and the actin cytoskeleton wasvisualized by fluorescence microscopy after incubation with phalloidin-Alexa488. Representative en face views of MTAL epithelium are shown. MTAL cellsincubated with hyperosmotic NaCl in the absence of drugs ( a ) exhibita thin cortical F-actin ring (filled arrow) and a dense cytoplasmic network ofF-actin bundles (dashed arrow). Jasplakinolide ( b ) induced a cleardensification of the cellular F-actin network with diffuse punctuatedF-actin-rich structures (actin clumps). MTAL cells incubated with swinholide A( c ) exhibited a dense cortical F-actin ring and a sparse diffuseF-actin network, similarly to MTAL cells incubated under isotonic conditions(see Fig. 2 ). Latrunculin B( d ) induced disorganization of the actin cytoskeleton with partialdisruption of the cortical F-actin ring.2 e/ j/ g2 t$ w7 N6 V5 R
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The role of actin polymerization or depolymerization and of the generationof new actin filaments of cellular volume recovery was assessed usingactin-interfering drugs. Figure4 A shows that jasplakinolide, which induces actinpolymerization and prevents actin depolymerization, almost completelyprevented the partial recovery of the initial cellular volume observed afterincubation of isolated MTALs for 30 min under hypertonic conditions but in theabsence of drug (% of initial volume; NaCl: 88.48 ± 4.71; NaCl  jasplakinolide: 74.65 ± 0.71; P Fig. 4 B ), whichprevents the generation of new actin filaments, increased cell shrinkage after5-min incubation under hypertonic conditions (% of initial volume; NaCl: 69.61± 3.90; NaCl swinholide: 51.85 ± 4.86; P of the initial cellular volume observedafter 30 min in the presence of hyperosmotic NaCl (% of initial volume; NaCl:88.48 ± 4.71; NaCl swinholide: 75.90 ± 3.09; P 0.05; Fig. 4 B ).Finally, latrunculin B, which depolymerizes the actin cytoskeleton, largelyattenuated cell shrinkage in response to 5-min incubation with hyperosmoticNaCl (% of initial volume; NaCl: 69.61 ± 3.90; NaCl latrunculin:80.84 ± 4.28; P cellular volume after 30-min incubation (% of initial volume; NaCl: 88.48± 4.71; NaCl latrunculin: 102.54 ± 4.94; P Fig. 4 C ). Thus, in ratMTALs, inhibition of actin depolymerization and generation of new actinfilaments decrease the efficacy of RVI, whereas actin depolymerization potentiates cell volume recovery after an acute hypertonic challenge.

Fig. 4. Effect of jasplakinolide, swinholide A, and latrunculin B on cellularvolume in response to extracellular hypertonicity. Microdissected MTALs wereincubated under isosmotic ( time 0 ) or hyperosmotic (600 mosM/l for 1to 30 min) conditions, without or with addition of the followingactin-interfering drugs: jasplakinolide ( A ), swinholide A( B ), and latrunculin B ( C ). Cellular volume was determinedfrom photographs. Results are expressed as a percentage of control values andare means ± SE from 5 independent experiments (* P
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Interfering with actin polymerization and remodeling did not alter MAPKactivation by hyperosmotic NaCl. The role of actin cytoskeletonremodeling in MAPKs activation in response to extracellular hypertonicty wasassessed by measurement of the phosphorylation level of ERK and p38 kinase inthe absence or presence of actin-interfering drugs. Figure 5 shows that theincreases in phosphorylation levels of ERK and p38 kinase observed afterincubation at 37°C for 10 min under hypertonic conditions in the presenceof jasplakinolide, or swinholide A or latrunculin B, were similar to thoseinduced by hyperosmotic NaCl alone. Similarly, ERK and p38 kinasephosphorylation levels were not altered by actin-interfering drugs in MTALsincubated under isotonic conditions (data not shown). Therefore, activation ofERK and p38 kinase in response to extracellular hypertonicity is independentof actin cytoskeleton remodeling.' t1 l( {$ |5 n3 J7 _! _3 W9 h
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Fig. 5. Effect of jasplakinolide (Jas), swinholide A (Swi), and latrunculin B (Lat)on ERK and p38 kinase phosphorylation levels in response to extracellularhypertonicity. MTAL suspensions were incubated under isosmotic (Iso; openbars) or hyperosmotic conditions (600 mosM/l, filled bars) with addition ofNaCl in the absence [vehicle (Veh)] or presence of 100 µg/ml Lat, 10 µMJas, or 50 µM Swi. The phosphorylation levels of p38 kinase ( A )and ERK ( B ) were measured by Western blot analysis. Afterdensitometric quantification, results were expressed as a percentage ofcontrol values and are means ± SE from 4 independent experiments(* P A and B, top :representative Western blot analyses.

p38 Kinase was involved in cellular volume recovery and actin cytoskeleton reorganization following extracellular hypertonic challenge. The following experiments were designed to study the role of MAPKs in cellularvolume variations and actin cytoskeleton reorganization induced byextracellular hypertonicity. Inhibition of the ERK signaling pathway by4.10 - 4 M PD-98059 (Calbiochem) modified neithercellular volume variation nor TX-100-insoluble/soluble actin ratio profiles inresponse to hyperosmotic NaCl (data not shown). Inhibition of p38 kinase by10 - 5 M SB-203580 (Calbiochem) slightly increasedthe maximal extent of hypertonicity-induced cell shrinkage observed after 10min (% of initial volume; NaCl: 77.14 ± 2.82; NaCl SB: 65.51 ±2.21 ± 4.94; P recovery after 30 min (% of initial volume; NaCl: 91.28 ± 1.21;NaCl SB: 78.05 ± 3.11; P Fig. 6 A ). In addition,SB-203580 attenuated the early increase in proportion to TX-100-insolubleactin observed after 2 min in the presence of hyperosmotic NaCl(TX-100-insoluble/TX-100-soluble actin; NaCl: 2.57 ± 0.43; NaCl SB:1.79 ± 0.25; P of TX-100-insoluble actin observed after 30-min incubation with hyperosmotic NaCl (TX-100-insoluble/TX-100-soluble actin; NaCl: 2.46 ±0.30; NaCl SB: 1.50 ± 0.09; P Fig. 6 B ). Similarresults were obtained in the presence of hyperosmotic sucrose (data notshown). As previously shown ( 35 ), SB-203580 did not alterMTAL cellular volume measured under isotonic conditions. Thus both cellularvolume recovery and sustained actin polymerization phase, which occurconcomitantly, are dependent on p38 kinase activity.
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Fig. 6. Role of p38 kinase on regulatory volume increase and actin cytoskeletonremodeling in response to extracellular hypertonicity. Microdissected MTALs( A ) or MTAL suspensions ( B ) were incubated under isosmotic( time 0 ) or hyperosmotic (600 mosM/l for 1 to 30 min) conditions,without or with addition of 10 - 5 M of SB-203580. A : cellular volume was determined from photographs. Results areexpressed as a percentage of control values and are means ± SE from 11independent experiments (* P B : actinsorting between TX-100-insoluble and -soluble fractions. TheTX-100-insoluble/soluble actin ratio was determined as in Fig. 1 and results are means± SE from 8 independent experiments (* P -actinsorting in the absence or presence of SB-203580 is shown.

Inhibition of p38 kinase activity and swinholide A both prevent F-actinredistribution in response to extracellular hypertonicity. SB-203580,which inhibits p38 kinase, and swinholide A, which prevents generation of newactin filaments, both inhibited the sustained actin polymerization anddecreased the efficacy of RVI in response to extracellular hypertonicity. Wetherefore assessed by fluorescence microscopy the effect of SB-203580 andswinholide A on F-actin redistribution following hypertonic challenge. Asshown by Fig. 7, SB-203580 andswinholide A strongly attenuated the hypertonicity-induced redistribution ofF-actin from the dense cortical F-actin ring to the diffuse network of F-actinbundles (compare Fig. 7, A and B ), compared with tubules incubated in the presence ofhyperosmotic NaCl alone (compare Fig. 7, B - D ). These results suggest that SB-203580 andswinholide A share the same mechanism of inhibition of actin cytoskeletonremodeling.
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Fig. 7. Effect of p38 kinase inhibition and swinholide A on actin cytoskeletonreorganization induced by extracellular hypertonicity. MTAL suspensions wereincubated for 30 min under isosmotic ( A ) or hyperosmotic conditions(600 mosM/l) with addition of NaCl ( B, C, and D ),without or with addition of 10 - 5 M SB-203580( C ) or 50 µM swinholide A ( D ). Tubules were then fixedwith 4% paraformaldehyde, permeabilized by 0.1% TX-100, and the actincytoskeleton was visualized by fluorescence microscopy after incubation withphalloidin-Alexa 488. Representative en face views of MTAL epithelium areshown. After incubation under hypertonic conditions ( B ), MTAL cellsexhibit a thin cortical F-actin ring (filled arrow) and a dense diffusenetwork of F-actin bundles (dashed arrow). The hypertonicity-inducedredistribution of F-actin from the cortical ring to the diffuse network wasprevented by SB-203580 ( C ) and swinholide A ( D ).% h& s0 e& T! O- t
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DISCUSSION7 q6 v' ?  x( \% s( Y

The present study demonstrates that extracellular hypertonicity inducedactin cytoskeleton remodeling in native rat MTAL cells. Hypertonicity-inducedremodeling of the actin cytoskeleton was dependent on p38 kinase activity andparticipated with the RVI. Results suggest that both cortical F-actindepolymerization and build-up of a diffuse F-actin network facilitate RVI,most likely through modulation of ion transporter activity.

Rat MTAL cells, which are physiologically exposed to large variations ininterstitial osmolality ( 23 ),exhibit a polyphasic actin polymerization profile in response to extracellularhyperosmolality (see Fig. 1 ).The initial rapid actin polymerization and depolymerization phases were notassociated with detectable actin filament redistribution and were shared byhyperosmotic challenges induced by NaCl, sucrose, and urea. Because ureaaltered neither cellular volume nor MAPK activity( 35 ), the transient phases ofactin polymerization-depolymerization are obviously independent of cellshrinkage and MAPK activation. In whole organisms, the interstitial osmolalityof the kidney medulla increases progressively under antidiuresis conditions, whereas under ex vivo experimental conditions used in this study, extracellular osmolality increased abruptly. We therefore cannot exclude thepossibility that the observed rapid changes in the levels of actinpolymerization observed during the first 10 min of incubation are due to anacute increase in intracellular osmolality. Exposure of rat MTAL cells tohyperosmotic NaCl and sucrose, but not urea, induced a progressive actinpolymerization phase (from 10- to 30-min incubation) and redistribution of F-actin from a dense cortical ring to a diffuse network of F-actin bundlesthat may rely on cell shrinkage and subsequent MAPK activation( 35 ). Actin polymerization andredistribution of F-actin were more pronounced in response to hyperosmoticsucrose, which decreases cellular volume and activates MAPKs to a larger extent than NaCl. In contrast, the sustained phase of actin polymerization andthe redistribution of F-actin were not observed in the presence ofhyperosmotic urea, which does not alter cell volume and MAPK activity. Theabolition of sustained actin polymerization and redistribution of F-actin by aspecific p38 kinase inhibitor further support this interpretation (see Figs. 6 and 7 ).! x5 t4 Z8 ~  \% }
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Our results suggest that actin cytoskeleton remodeling is dependent on p38kinase activation (see Figs. 6 and 7 ). The p38kinase-dependent actin cytoskeleton remodeling may be mediated, at least in part, through phosphorylation of HSP25/27, a small heat shock protein thatmodulates actin polymerization( 27 ). Phosphorylated HSP25/27promotes actin polymerization, whereas its nonphosphorylated form isinhibitory ( 2, 8, 25, 31, 38 ). In intact cells, activated p38 kinase phosphorylates and activates MAPKAP kinase 2/3, which inturn phosphorylates HSP25/27( 17, 28, 37 ) and thereby promotesredistribution of HSP25/27 from the cytoplasm to the actin cytoskeleton( 52 ). However, in addition tothe MAPK pathway, cell shrinkage increases tyrosine phosphorylation of asubset of proteins including nonreceptor tyrosine kinases andcytoskeleton-associated proteins( 20, 21, 24 ). Therefore, the tyrosinekinase pathway may also participate in the actin cytoskeleton remodelinginduced by extracellular hypertonicity.# a1 [( ?6 W: \
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In addition to native MTAL cells, remodeling of the actin cytoskeleton inresponse to extracellular hyperosmolality has been observed in yeast( 12 ), Dictyostelium ( 53 ), and cultured mammalian nonepithelial cells ( 13, 18, 19, 32 ) as well as in epithelial Madin-Darby canine kidney cells( 39 ). Results of the present study and from the literature indicate that actin cytoskeleton remodelingexhibits some degree of cell specificity. In native rat MTAL cells (see Fig. 2 ) and glial cells( 32 ), hypertonicity inducedredistribution of F-actin from the cortical ring to a diffuse network of actinbundles, whereas in fibroblasts and HL60 cells, a densification of theperipheral actin ring was observed( 13, 18 ). Moreover, the sustainedactin polymerization phase observed in native rat MTAL cells was absent incultured HL60 cells ( 19 ).These different patterns of actin cytoskeleton remodeling are associated withdifferences in RVI efficacy. Indeed, in contrast to the majority of cellsexhibiting little or no RVI, MTAL cells undergo robust RVI( 35, 43 ).
# \  o+ Z% k8 j1 c9 W+ S
The temporal relationship between RVI and actin cytoskeleton reorganization, taken together with results obtained with actin-interfering drugs, suggests that both cortical F-actin depolymerization and de novo actinpolymerization resulting in the generation of a diffuse network of F-actinbundles play an important role in the RVI of MTAL cells. Results of thepresent study indicate that whole cell actin depolymerization with latrunculinB facilitates RVI, whereas global inhibition of actin depolymerization by jasplakinolide antagonizes RVI (see Figs. 3 and 4 ). These results suggest thatdepolymerization of F-actin is required for RVI in MTAL epithelial cells. Inaddition, fluorescence microscopy imaging shows that RVI is associated withreduced cortical F-actin staining (see Fig.2 ), suggesting that the F-actin depolymerization process involvedin RVI specifically takes place at the level of the cortical F-actin ring inMTAL epithelial cells. This result contrasts with those obtained innonepithelial cells (HL60) that exhibit a densification of the corticalF-actin ring in response to hypertonicity but which do not undergo RVI( 13, 18 ). On the other hand, our results show that swinholide A or SB-203580, an inhibitor of p38 kinase,prevented the hypertonicity-induced generation of diffuse F-actin bundles andreduced the efficacy of RVI (see Figs. 4 and 6 ). These results suggest that,in addition to cortical F-actin ring depolymerization, the generation of adense diffuse network of F-actin bundles facilitates RVI. At fist glance, thisfinding contrasts with the effect of jasplakinolide, which increases the actinpolymerization level and prevents the RVI. It should be noticed, however, thatjasplakinolide also increased actin polymerization at the level of thecortical F-actin ring, an effect that most likely antagonizes RVI.

It is well established that RVI is associated with ion transporter activation including Na-K-2Cl cotransporter, Na/H exchanger, Cl/HCO 3 exchanger, and Na-K-ATPase( 4, 15, 29, 43, 47 ), which might, at least inpart, be dependent on cortical actin polymerization level. For instance,inhibition of F-actin depolymerization by phalloidin or jasplakinolide impairsthe activation of the Na-K-2Cl cotransporter by cAMP in MTAL cells( 49 ). Conversely, depolymerization of F-actin by cytochalasin D stimulates Na-K-2Cl cotransporter in intestinal cells( 30 ). Actin polymerization maycontrol the activity of ion transporters in different ways. A shift inF-actin-G-actin equilibrium toward G-actin may stimulate the activity ofspecific ion transporters, as shown for Na-K-ATPase ( 9 ) and epithelial Na channels( 3, 10 ). On the other hand, depolymerization of the cortical F-actin ring may promote the exocytosis ofion, solute, and water transporters as demonstrated for the Na/H exchangerNHE3 ( 11 ), volume-sensitiveCl - channels( 33 ), the glucose transporterGLUT4 ( 26 ), and the waterchannel aquaporin-2 ( 22 ). Theresults of the present study indicate that, at the level of the whole cell,the equilibrium between actin polymerization and depolymerization is shifted toward actin polymerization during RVI (see Fig. 1 ). This polymerization process results in the generation of a dense and diffuse network of F-actinbundles (see Fig. 2 ) that mayplay a functional role in the defense against cell shrinkage. Indeed,inhibition of the sustained actin polymerization phase and the densification of the diffuse F-actin network by swinholide A and SB-203580 both increasedthe extent of maximal cell shrinkage and reduced the efficacy of RVI inresponse to hypertonicity (see Figs. 4, 6, and 7 ). This effect might be partlyachieved through mechanical constraints exerted on the cell membrane, asdescribed for lamellipodia or filipodia formation( 44 ). It occurs, however, mostlikely indirectly via spacial control of signaling events and/or facilitationof the delivery of ion transporters from intracellular stores to the plasmamembrane, as shown for the GLUT4 glucose transporter in response to insulin( 46 ).

Because activation of MAPKs is mediated by cell shrinkage in rat MTAL cells( 35 ), the hypothesis that theactin cytoskeleton may be part of the osmosensing machinery was considered.Our results, however, suggest that actin cytoskeleton remodeling and integrityare not essential for the activation of MAPKs in response to increasedextracellular osmolality. Indeed, interfering with neither the polymerizationlevel of actin nor with the generation of new actin filaments decreased the extent of ERK and p38 kinase activation in response to extracellular hypertonicity (see Fig. 5 ).Therefore, alternative mechanisms such as an increase in cytoplasmicconcentration of macromolecules ( 34 ) or aggregation of growthfactor and cytokine receptors leading to their ligand-independent activation( 36 ) have to beconsidered.$ a7 E5 U4 y/ Q* x8 Q& @
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In conclusion, we showed that an acute extracellular hypertonic challengeinduces actin cytoskeleton remodeling consisting of F-actin redistributionfrom a cortical ring to a diffuse network of F-actin bundles in native ratMTAL cells. We propose the following working hypothesis summarized by Fig. 8. Cell shrinkage inducesp38 kinase activation, which in turn, promotes cortical F-actin ringdepolymerization and generation of a dense diffuse network of F-actin bundlesthat both promote RVI most likely through modulation of ion transporteractivity. Further investigation is required to identify the molecular playersinvolved in actin cytoskeleton remodeling. In addition, the role of actincytoskeleton remodeling in the control of the activity and/or abundance of iontransporters involved in RVI remains to be determined.
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Fig. 8. Schematic representation of the sequence of events linking p38 kinaseactivation, actin cytoskeleton, and regulatory volume increase (RVI) in MTALepithelial cells. Exposure of MTAL epithelial cells to extracellularhypertonicity induces cell shrinkage leading to the activation of p38 kinase.Increased p38 kinase activity promotes depolymerization of the corticalF-actin ring and polymerization of new actin filaments generating a densediffuse network of F-actin bundles. Both processes may stimulate the activityof ion transporters leading to intracellular ion accumulation, secondary waterinflux, and recovery of the initial cellular volume.
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DISCLOSURES9 R7 g) E. t2 j( K  P; U

This work was supported in part by Grants 31-50-643.97 and31-56830.99 from the Swiss National Foundation to E. Féraille andby a grant from the Fondation Novartis pour la Recherche en SciencesMédico-biologiques to E. Féraille.6 B7 \5 r$ G; p7 _4 Q7 @: V/ x' X
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ACKNOWLEDGMENTS

We thank Dr. C. Chaponnier for helpful discussions and critical reading ofthe manuscript.& h% N7 Z& l# f$ K
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