Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton remodeling in rat kidney MTAL

Mauro Bustamante,1 Frank Roger,1 Marie-Luce Bochaton-Piallat,2 Giulio Gabbiani,2 Pierre-Yves Martin,1 and Eric Féraille1

1Division de Néphrologie, Fondation pour Recherches Médicales, and 2Département de Pathologie, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland

Submitted 7 January 2003 ; accepted in final form 22 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The kidney medulla is physiologically exposed to variations in extracellular osmolality. In response to hypertonic cell shrinkage, cells of the rat kidney medullary thick ascending limb of Henle's loop undergo p38 kinase-dependent regulatory volume increase (RVI). In the present study, we investigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases and reduces cellular volume, induced a sustained actin polymerization occurring after 10 min and concurrently with RVI. In contrast, hyperosmotic urea, which does not modify MAP kinase activity and cellular volume, did not induce sustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution of F-actin from a dense cortical ring to a diffuse network of actin bundles. Stabilization of actin filaments by jasplakinolide and inhibition of the generation of new actin filaments by swinholide A prevented RVI, whereas depolymerization of actin filaments by latrunculin B attenuated cell shrinkage and 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 abolished sustained actin polymerization, actin redistribution, and decreased RVI efficacy. We therefore propose that in rat kidney the medullary thick ascending limb of Henle's loop exposed to extracellular hypertonicity, p38 kinase activation induces depolymerization of the F-actin cortical ring and polymerization of a dense diffuse F-actin network that both contribute to increase RVI efficacy.

mitogen-activated protein kinase; osmolarity; kidney medulla; cell volume


DURING DIURESIS AND antidiuresis, the kidney medulla of vertebrates is exposed to large fluctuations in interstitial osmolality (23), which challenges cell volume constancy. Under antidiuresis, the countercurrent concentration mechanism initiated by active NaCl reabsorption by the medullary thick ascending limb of Henle's loop (MTAL) leads to NaCl and urea accumulation in the renal medulla. MTAL cells are therefore of special interest because they have developed adaptive mechanisms to survive and function in a hypertonic environment. The high interstitial osmolality induces changes in the activity of solute transporters and enzymes involved in solute accumulation and in the expression of genes encoding enzymes required for solute synthesis, stress resistance, and yeast cell wall structure (34). After cell shrinkage in response to extracellular hypertonicity, MTAL cells progressively recover their initial volume through regulatory volume increase (RVI). This process occurs within minutes and is mediated by stimulation of ion transporters that increase intracellular ionic concentrations, which drive water influx and restore initial cellular volume (34, 43). The rapid increase in ionic concentrations is followed by a slow accumulation of intracellular compatible osmolytes, such as sorbitol, myoinositol, taurine, betaine, and glycerophosphocholine, allowing recovery of normal ionic concentrations (7). This process is a long-term mechanism occurring within hours or days and counteracts increased extracellular osmotic pressure.

Hypertonic conditions activate MAPK, which is an important signal transducer linking signals from the cell surface to the nucleus. MAPKs are serine/threonine kinases activated by a cascade of kinases involving two upstream kinases, MAPKKK and MAPKK (48). In mammalian cells, MAPKs are divided into three families, each responding to distinct extracellular stimuli: ERK 1 and 2, JNK (also known as stress-activated protein kinases 1), and p38 kinases (or stress-activated protein kinase 2). In our previous study, we showed that cell shrinkage, rather than intracellular hypertonicity, triggers the activation of ERK and p38 kinase in rat MTALs (35). MAPK activation levels were dependent on the osmolyte used to increase extracellular osmolality. Hyperosmotic NaCl induced cell shrinkage and activated ERK and p38 kinase but not JNK. In comparison, hyperosmotic sucrose induced even greater cell shrinkage and stronger activation of ERK and p38 kinase and also activated JNK but to a lesser extent. By contrast, hyperosmotic urea altered neither cell volume nor MAPK activity. Both hypertonic NaCl and sucrose triggered cellular RVI that restored, almost completely for NaCl and partially for sucrose, the initial cellular volume. Inhibition of p38 kinase decreased the efficiency of RVI, implying a major role of this kinase in this process, whereas inhibition of ERK did not alter RVI.

Modifications of cellular architecture related to hypertonicity-induced cell shrinkage are associated with a reorganization of the architecture of the actin 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 cascades leading to RVI. In addition, signal transduction cascades leading to remodeling of the actin cytoskeleton and to MAPK activation share some common elements. For instance, small G proteins of the Rho family, such as Cdc42 and Rac 1, are involved in both actin cytoskeleton remodeling through filipodia and lamellipodia formation (45) and in signaling events leading to p38 kinase activation (1, 51). Activation of p38 kinase may, in turn, control actin cytoskeleton dynamics through the activation of downstream kinases such as MAPKAP kinase 2/3 or PRAK, which phosphorylate HSP 25/27 (17, 28, 37), a small heat shock protein that modulates actin polymerization (27). The actin cytoskeleton may also control the activity of ion transporters, leading to intracellular NaCl uptake and secondary water influx, either directly, through F-actin/G-actin ratio dynamics (9, 10), or indirectly, through binding of signaling modules (50) and/or modulation of endocytotic-exocytotic events (42). This study was therefore undertaken to investigate the relationship between actin cytoskeleton remodeling and RVI in rat MTAL.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of single MTALs. Male Wistar rats weighing 150–200 g were anesthetized with pentobarbital sodium (5 mg/100 g body wt ip), and the left kidney was immediately removed after perfusion with ice-cold incubation solution (120 mM NaCl, 5 mM RbCl, 4 mM NaHCO3, 1 mM CaCl2, 1 mM MgSO4, 0.2 mM NaH2PO4, 0.15 mM Na2HPO4, 5 mM glucose, 10 mM lactate, 1 mM pyruvate, 4 mM essential and nonessential amino acids, 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 incubation solution (see above) containing 0.05% (wt/vol) collagenase, kidney slices were stored at 4°C, and single MTALs were microdissected under stereomicroscopic control in oxygenated (95% O2-5% CO2) incubation solution.

Preparation of MTAL suspensions. The two kidneys were perfused with ice-cold incubation solution without collagenase. The inner stripes of the outer medulla were excised, minced on ice, and fragments of medullary tubules were obtained by gentle pressure through nylon filters with a pore size decreasing from 150 to 100 µm. After centrifugation, the pellet was resuspended in ice-cold oxygenated (95% O2-5% CO2) incubation solution. As controlled under a stereomicroscope, MTALs account for ~90% of the tubule fragments in this preparation. Therefore, it will be referred as MTAL suspension.

Determination of the Triton X-100-soluble and -insoluble actin fractions. Estimation of actin polymerization level was performed by determining the Triton X (TX)-100-soluble/TX-100-insoluble actin ratio. Indeed, it is largely admitted that F-actin, i.e., polymerized actin, is contained in the TX-100-insoluble fraction and that G-actin, i.e., monomeric actin, is contained in the TX-100-soluble fraction (16). After 1-h preincubation at 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 or hyperosmotic (addition of 300 mosM/l NaCl, sucrose, or urea) conditions. Incubation was stopped by cooling and centrifugation at 6,000 g for 5 min at 4°C. The pellet was saved and 20 µl of ice-cold lysis buffer (20 mM Tris · HCl, 2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mM Na4O7P2, 2 mM Na3VO4, 1 mM AEBSF, 10 µg/ml leupeptin, 4 µg/ml aprotinin, 1% Triton X-100, pH 7.45) were added. After 5 min of centrifugation at 12,000 g, the supernatant was saved, the pellet was then mixed with fresh lysis buffer, and after a centrifugation step at 12,000 g, the second supernatant was saved. The final pellet was then suspended in sample buffer and an equal volume of sample buffer was added to the pooled supernatants. The proteins from pooled supernatants and pellet were then separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Waters, MA), and {beta}-actin was detected by immunoblot using a monoclonal anti-{beta}-actin antibody (AC-15, Sigma, St. Louis, MO) at 1:40,000 dilution (vol/vol). After incubation with anti-mouse IgG coupled to horseradish peroxidase (Transduction Laboratories, Lexington, UK) at 1:10,000 dilution (vol/vol), immunoreactivity was detected by chemiluminescence using the Super Signal Substrate method (Pierce, Rockford, IL). Results were quantified under conditions of linearity by integration of the density of total area of each band using a video densitometer and Image-Quant software (Molecular Dynamics, Sunnyvale, CA). Results are expressed as a percentage of the control optical density (isotonic medium) and are means ± SE.

Determination of the phosphorylation level of ERK and p38 kinase. After 1-h preincubation at 30°C in isotonic incubation solution with or without addition of drugs, MTAL suspensions were incubated at 37°C for 10 min under isosmotic or hyperosmotic (addition of 300 mosM/l NaCl) conditions. Incubation was stopped by cooling and centrifugation at 6,000 g for 5 min at 4°C. After addition of lysis buffer, protein content was measured by 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-P and 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 1:10,000 dilution (vol/vol), immunoreactivity was detected by chemiluminescence, and results were quantified and expressed as described above.

Determination of MTAL cellular volume. A pool of three isolated MTALs was transferred into the concavity of a bacteriological slide coated with dried BSA. After 1-h preincubation at 30°C in isosmotic incubation solution with or without addition of drugs, MTALs were incubated at 37°C for 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 with or without drugs. MTALs were visualized with an inverted microscope, and photographs of the same tubules were taken at the end of the preincubation period and after incubation. MTAL volume (V) was calculated from the measured radius (R) and length (L) of the tubules at a 1,000-fold magnification using the formula V = {pi}R2 x L. Because the lumen is collapsed in nonperfused tubules, we assumed that MTAL volume measurement is an appropriate estimate of MTAL cellular volume. Results are expressed as a percentage of the control volume (end of the preincubation period) and are means ± SE.

Fluorescence microscopy. MTAL suspensions were preincubated at 30°C for 1 h with or without drugs and then incubated at 37°C under isosmotic 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 fixed with 3.7% paraformaldehyde for 10 min at room temperature. After three washes in PBS, fixed tubules were permeabilized with 0.1% Triton X-100 for 1 min at room temperature. After a new series of three washes in PBS, specimens were incubated 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-immersion plan-neofluar x40:1.3 objective. Images were acquired with a high-sensitivity, high-resolution color camera (Axiocam, Carl Zeiss). Pictures were printed with a digital pictrography 4000 printer (Fujifilm, Tokyo, Japan).

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 kinase immunoreactivity was done using the Kruskall-Wallis test. P values <0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Actin cytoskeleton remodeling in response to hyperosmotic NaCl, sucrose, or urea. We previously showed that hyperosmotic NaCl and sucrose, but not urea, induced both MAPK activation and cell shrinkage in rat MTAL cells (35). Because cell shrinkage and/or intracellular hypertonicity may induce remodeling of the actin cytoskeleton, we compared the time course of the effects of hyperosmotic NaCl, sucrose, and urea on the F-actin/G-actin ratio and cellular volume in rat MTALs.

Figure 1 shows that increasing 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 its basal level after 10-min incubation. These rapid variations in cellular TX-100-insoluble actin content were followed by a progressive increase in proportion of TX-100-insoluble actin above the basal levels in samples incubated 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 < 0.05). This increase in proportion of TX-100-insoluble actin was sustained for at least 60 min (data not shown). The progressive increase in proportion of TX-100-insoluble actin was more pronounced 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 < 0.05) compared with hyperosmotic NaCl (Fig. 1, A and B). In contrast, for incubation periods ranging from 10 to 30 min, hyperosmotic urea did not induce significant variations in the TX-100-insoluble/TX-100-soluble actin ratio (isotonic 30 min: 1.75 ± 0.11; urea 30 min: 1.60 ± 0.12; not significant; Fig. 1C). As shown previously (35), both hyperosmotic NaCl and sucrose rapidly induced cell shrinkage with a maximal decrease in cellular volume observed after 10 min of incubation (% of initial cellular volume; NaCl: 68.98 ± 2.21; sucrose: 65.47 ± 0.58). After 30-min incubation in the presence of hyperosmotic NaCl or sucrose, a partial 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. 1C). These results show that, in MTAL cells, acute extracellular hyperosmolality induces polyphasic actin cytoskeleton remodeling reflected by the observed changes in the F-actin/G-actin ratio. However, sustained actin polymerization reflected by the progressive increase in cellular F-actin content was only observed in response to osmolytes inducing cell shrinkage and this event occurred concomitantly with the partial recovery of the initial cellular volume.



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Fig. 1. Effect of hyperosmotic NaCl, sucrose, and urea on actin polymerization level 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. {beta}-Actin sorting between Triton (TX)-100-insoluble (i) and -soluble (s) fractions was analyzed by Western blot analysis and the TX-100-insoluble/soluble actin ratio was calculated after quantification by densitometry. Cellular volume was determined from photographs. Results are means ± SE from 6 to 11 independent experiments (*P < 0.05 vs. time 0). The graphs show the time course of TX-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 analyses illustrating actin sorting after 30-min incubation in the presence of hyperosmotic NaCl, hyperosmotic sucrose, or hyperosmotic urea.

 

The effect of extracellular hyperosmolality on actin cytoskeleton organization was assessed by fluorescence microscopy on isolated rat MTALs incubated at 37°C for 30 min under isosmotic or hyperosmotic conditions. As shown by Fig. 2A, rat MTAL cells incubated under isosmotic conditions exhibited a dense cortical F-actin ring delineating the cell periphery and a sparse diffuse network of F-actin bundles. After exposure of MTALs to hyperosmotic NaCl, the cortical F-actin ring was thinner and the diffuse F-actin network was more developed compared with tubules incubated under isosmotic conditions (Fig. 2B). This redistribution of F-actin was more pronounced after incubation of tubules in the presence of hyperosmotic sucrose (Fig. 2C). In contrast, hyperosmotic urea did not induce any significant change in F-actin distribution (Fig. 2D). Therefore, the sustained actin polymerization phase observed in response to hyperosmotic NaCl and sucrose was associated with a redistribution of F-actin from the cortical F-actin ring to a diffuse network of F-actin bundles. In contrast, the early actin polymerization phase was not associated with apparent F-actin redistribution (data not shown).



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Fig. 2. Effect of hyperosmotic NaCl, sucrose, and urea on actin cytoskeleton organization. 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 then fixed with 4% paraformaldehyde, permeabilized by 0.1% TX-100, and Factin was visualized by fluorescence microscopy after incubation with phalloidin-Alexa 488. 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 diffuse network of F-actin bundles, whereas hyperosmotic urea (D) did not alter actin cytoskeleton morphology.

 

Interfering with actin polymerization and remodeling altered cellular volume recovery under hypertonic conditions. The actin cytoskeleton is a highly dynamic structure that undergoes constant remodeling, consisting of spatially and temporally regulated polymerization and depolymerization of preexisting filaments as well as nucleation and branching of new actin filaments. New pharmacological tools derived from marine sponges (40) were used to study the role of actin polymerization-depolymerization and generation of new actin filaments on cellular volume recovery under hypertonic conditions. Jasplakinolide binds to both ends of actin filaments preventing their depolymerization and also causes rapid nucleation of actin polymerization (5, 40). As expected from its mechanism of action, 10 µM jasplakinolide (Calbiochem, San Diego, CA) increased the proportion of TX-100-insoluble actin in tubules incubated under hypertonic conditions for 30 min (TX-100-insoluble/TX-100-soluble actin; NaCl: 2.87 ± 0.49; NaCl + jasplakinolide: 4.02 ± 0.45; P < 0.05; Fig. 3A). In addition, fluorescence microscopy revealed that jasplakinolide-treated tubules exhibited a very dense F-actin network with diffuse thick and short actin-rich structures, i.e., actin clumps, throughout the cytoplasm (Fig. 3B). Therefore, jasplakinolide efficiently increases the actin polymerization in rat MTAL cells. Swinholide A inhibits actin filament nucleation and elongation (6, 40), thereby preventing stimulus-induced actin polymerization without affecting the intact actin network (46). Measurement of the partition of actin between TX-100-soluble and -insoluble fractions showed that, 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. 3A). Fluorescence microscopy, however, showed that reorganization of the actin cytoskeleton induced by hyperosmotic NaCl was largely prevented by swinholide A (compare a and c, Fig. 3B). Indeed, most F-actin remained in the cortical ring and the density of the diffuse F-actin network was unchanged compared with MTALs incubated under isotonic conditions. Therefore, swinholide A does not significantly alter actin cytoskeleton organization but prevents its hypertonicity-induced remodeling in rat MTAL cells. Latrunculin B sequesters monomeric actin and decreases G-actin availability, resulting in actin filament depolymerization (40, 41). Consistent with its actin-depolymerizing properties, 100 µg/ml latrunculin B (Calbiochem) induced a large decrease in proportion to TX-100-insoluble actin with respect to 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. 3A). In addition, fluorescence microscopy revealed that latrunculin B disorganized the actin cytoskeleton. The cortical F-actin ring became irregular and discontinuous, and the diffuse F-actin network was almost completely disrupted (compare a and d, Fig. 3B). Therefore, latrunculin B potently depolymerizes the actin cytoskeleton in rat MTAL cells.



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Fig. 3. Effect of jasplakinolide, swinholide A, and latrunculin B on actin polymerization level and actin cytoskeleton reorganization in response to extracellular hypertonicity. MTAL suspensions were incubated under hyperosmotic (600 mM NaCl) conditions without (control; H) or with 10 µM jasplakinolide, 50 µM swinholide A, or 100 µg/ml latrunculin B. A: actin sorting between TX-100-insoluble and -soluble fractions. The TX-100-insoluble/soluble actin ratio was determined as described in the legend of Fig. 1 and results are means ± SE from 3 independent experiments (*P < 0.05 vs. H). A representative Western blot analysis depicting {beta}-actin sorting in the absence or presence of drugs is shown. B: fluorescence imaging of the actin cytoskeleton. After 30-min incubation, tubules were fixed with 4% paraformaldehyde, permeabilized by 0.1% TX-100, and the actin cytoskeleton was visualized by fluorescence microscopy after incubation with phalloidin-Alexa 488. Representative en face views of MTAL epithelium are shown. MTAL cells incubated with hyperosmotic NaCl in the absence of drugs (a) exhibit a thin cortical F-actin ring (filled arrow) and a dense cytoplasmic network of F-actin bundles (dashed arrow). Jasplakinolide (b) induced a clear densification of the cellular F-actin network with diffuse punctuated F-actin-rich structures (actin clumps). MTAL cells incubated with swinholide A (c) exhibited a dense cortical F-actin ring and a sparse diffuse F-actin network, similarly to MTAL cells incubated under isotonic conditions (see Fig. 2). Latrunculin B (d) induced disorganization of the actin cytoskeleton with partial disruption of the cortical F-actin ring.

 

The role of actin polymerization or depolymerization and of the generation of new actin filaments of cellular volume recovery was assessed using actin-interfering drugs. Figure 4A shows that jasplakinolide, which induces actin polymerization and prevents actin depolymerization, almost completely prevented the partial recovery of the initial cellular volume observed after incubation of isolated MTALs for 30 min under hypertonic conditions but in the absence of drug (% of initial volume; NaCl: 88.48 ± 4.71; NaCl + jasplakinolide: 74.65 ± 0.71; P < 0.05). Swinholide A (Fig. 4B), which prevents the generation of new actin filaments, increased cell shrinkage after 5-min incubation under hypertonic conditions (% of initial volume; NaCl: 69.61 ± 3.90; NaCl + swinholide: 51.85 ± 4.86; P < 0.05) and decreased the extent of recovery of the initial cellular volume observed after 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. 4B). Finally, latrunculin B, which depolymerizes the actin cytoskeleton, largely attenuated cell shrinkage in response to 5-min incubation with hyperosmotic NaCl (% of initial volume; NaCl: 69.61 ± 3.90; NaCl + latrunculin: 80.84 ± 4.28; P < 0.05) and allowed a full recovery of cellular volume after 30-min incubation (% of initial volume; NaCl: 88.48 ± 4.71; NaCl + latrunculin: 102.54 ± 4.94; P < 0.05; Fig. 4C). Thus, in rat MTALs, inhibition of actin depolymerization and generation of new actin filaments decrease the efficacy of RVI, whereas actin depolymerization potentiates cell volume recovery after an acute hypertonic challenge.



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Fig. 4. Effect of jasplakinolide, swinholide A, and latrunculin B on cellular volume in response to extracellular hypertonicity. Microdissected MTALs were incubated under isosmotic (time 0) or hyperosmotic (600 mosM/l for 1 to 30 min) conditions, without or with addition of the following actin-interfering drugs: jasplakinolide (A), swinholide A (B), and latrunculin B (C). Cellular volume was determined from photographs. Results are expressed as a percentage of control values and are means ± SE from 5 independent experiments (*P < 0.05 vs. control).

 

Interfering with actin polymerization and remodeling did not alter MAPK activation by hyperosmotic NaCl. The role of actin cytoskeleton remodeling in MAPKs activation in response to extracellular hypertonicty was assessed by measurement of the phosphorylation level of ERK and p38 kinase in the absence or presence of actin-interfering drugs. Figure 5 shows that the increases in phosphorylation levels of ERK and p38 kinase observed after incubation at 37°C for 10 min under hypertonic conditions in the presence of jasplakinolide, or swinholide A or latrunculin B, were similar to those induced by hyperosmotic NaCl alone. Similarly, ERK and p38 kinase phosphorylation levels were not altered by actin-interfering drugs in MTALs incubated under isotonic conditions (data not shown). Therefore, activation of ERK and p38 kinase in response to extracellular hypertonicity is independent of actin cytoskeleton remodeling.



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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 extracellular hypertonicity. MTAL suspensions were incubated under isosmotic (Iso; open bars) or hyperosmotic conditions (600 mosM/l, filled bars) with addition of NaCl in the absence [vehicle (Veh)] or presence of 100 µg/ml Lat, 10 µM Jas, or 50 µM Swi. The phosphorylation levels of p38 kinase (A) and ERK (B) were measured by Western blot analysis. After densitometric quantification, results were expressed as a percentage of control values and are means ± SE from 4 independent experiments (*P < 0.05 vs. Veh). 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 cellular volume variations and actin cytoskeleton reorganization induced by extracellular hypertonicity. Inhibition of the ERK signaling pathway by 4.104 M PD-98059 (Calbiochem) modified neither cellular volume variation nor TX-100-insoluble/soluble actin ratio profiles in response to hyperosmotic NaCl (data not shown). Inhibition of p38 kinase by 105 M SB-203580 (Calbiochem) slightly increased the maximal extent of hypertonicity-induced cell shrinkage observed after 10 min (% of initial volume; NaCl: 77.14 ± 2.82; NaCl + SB: 65.51 ± 2.21 ± 4.94; P < 0.05) and decreased the efficacy of cell volume recovery after 30 min (% of initial volume; NaCl: 91.28 ± 1.21; NaCl + SB: 78.05 ± 3.11; P < 0.05; Fig. 6A). In addition, SB-203580 attenuated the early increase in proportion to TX-100-insoluble actin 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 < 0.05) and abolished the sustained increase in amounts 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 < 0.01; Fig. 6B). Similar results were obtained in the presence of hyperosmotic sucrose (data not shown). As previously shown (35), SB-203580 did not alter MTAL cellular volume measured under isotonic conditions. Thus both cellular volume recovery and sustained actin polymerization phase, which occur concomitantly, are dependent on p38 kinase activity.



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Fig. 6. Role of p38 kinase on regulatory volume increase and actin cytoskeleton remodeling 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 105 M of SB-203580. A: cellular volume was determined from photographs. Results are expressed as a percentage of control values and are means ± SE from 11 independent experiments (*P < 0.05 vs. control). B: actin sorting between TX-100-insoluble and -soluble fractions. The TX-100-insoluble/soluble actin ratio was determined as in Fig. 1 and results are means ± SE from 8 independent experiments (*P < 0.05 vs. control). A representative Western blot analysis depicting {beta}-actin sorting in the absence or presence of SB-203580 is shown.

 

Inhibition of p38 kinase activity and swinholide A both prevent F-actin redistribution in response to extracellular hypertonicity. SB-203580, which inhibits p38 kinase, and swinholide A, which prevents generation of new actin filaments, both inhibited the sustained actin polymerization and decreased the efficacy of RVI in response to extracellular hypertonicity. We therefore assessed by fluorescence microscopy the effect of SB-203580 and swinholide A on F-actin redistribution following hypertonic challenge. As shown by Fig. 7, SB-203580 and swinholide A strongly attenuated the hypertonicity-induced redistribution of F-actin from the dense cortical F-actin ring to the diffuse network of F-actin bundles (compare Fig. 7, A and B), compared with tubules incubated in the presence of hyperosmotic NaCl alone (compare Fig. 7, B-D). These results suggest that SB-203580 and swinholide A share the same mechanism of inhibition of actin cytoskeleton remodeling.



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Fig. 7. Effect of p38 kinase inhibition and swinholide A on actin cytoskeleton reorganization induced by extracellular hypertonicity. MTAL suspensions were incubated 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 105 M SB-203580 (C) or 50 µM swinholide A (D). Tubules were then fixed with 4% paraformaldehyde, permeabilized by 0.1% TX-100, and the actin cytoskeleton was visualized by fluorescence microscopy after incubation with phalloidin-Alexa 488. Representative en face views of MTAL epithelium are shown. After incubation under hypertonic conditions (B), MTAL cells exhibit a thin cortical F-actin ring (filled arrow) and a dense diffuse network of F-actin bundles (dashed arrow). The hypertonicity-induced redistribution of F-actin from the cortical ring to the diffuse network was prevented by SB-203580 (C) and swinholide A (D).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrates that extracellular hypertonicity induced actin cytoskeleton remodeling in native rat MTAL cells. Hypertonicity-induced remodeling of the actin cytoskeleton was dependent on p38 kinase activity and participated with the RVI. Results suggest that both cortical F-actin depolymerization 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 in interstitial osmolality (23), exhibit a polyphasic actin polymerization profile in response to extracellular hyperosmolality (see Fig. 1). The initial rapid actin polymerization and depolymerization phases were not associated with detectable actin filament redistribution and were shared by hyperosmotic challenges induced by NaCl, sucrose, and urea. Because urea altered neither cellular volume nor MAPK activity (35), the transient phases of actin polymerization-depolymerization are obviously independent of cell shrinkage and MAPK activation. In whole organisms, the interstitial osmolality of 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 the possibility that the observed rapid changes in the levels of actin polymerization observed during the first 10 min of incubation are due to an acute increase in intracellular osmolality. Exposure of rat MTAL cells to hyperosmotic NaCl and sucrose, but not urea, induced a progressive actin polymerization phase (from 10- to 30-min incubation) and redistribution of F-actin from a dense cortical ring to a diffuse network of F-actin bundles that may rely on cell shrinkage and subsequent MAPK activation (35). Actin polymerization and redistribution of F-actin were more pronounced in response to hyperosmotic sucrose, which decreases cellular volume and activates MAPKs to a larger extent than NaCl. In contrast, the sustained phase of actin polymerization and the redistribution of F-actin were not observed in the presence of hyperosmotic urea, which does not alter cell volume and MAPK activity. The abolition of sustained actin polymerization and redistribution of F-actin by a specific p38 kinase inhibitor further support this interpretation (see Figs. 6 and 7).

Our results suggest that actin cytoskeleton remodeling is dependent on p38 kinase activation (see Figs. 6 and 7). The p38 kinase-dependent actin cytoskeleton remodeling may be mediated, at least in part, through phosphorylation of HSP25/27, a small heat shock protein that modulates actin polymerization (27). Phosphorylated HSP25/27 promotes actin polymerization, whereas its nonphosphorylated form is inhibitory (2, 8, 25, 31, 38). In intact cells, activated p38 kinase phosphorylates and activates MAPKAP kinase 2/3, which in turn phosphorylates HSP25/27 (17, 28, 37) and thereby promotes redistribution of HSP25/27 from the cytoplasm to the actin cytoskeleton (52). However, in addition to the MAPK pathway, cell shrinkage increases tyrosine phosphorylation of a subset of proteins including nonreceptor tyrosine kinases and cytoskeleton-associated proteins (20, 21, 24). Therefore, the tyrosine kinase pathway may also participate in the actin cytoskeleton remodeling induced by extracellular hypertonicity.

In addition to native MTAL cells, remodeling of the actin cytoskeleton in response 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 remodeling exhibits some degree of cell specificity. In native rat MTAL cells (see Fig. 2) and glial cells (32), hypertonicity induced redistribution of F-actin from the cortical ring to a diffuse network of actin bundles, whereas in fibroblasts and HL60 cells, a densification of the peripheral actin ring was observed (13, 18). Moreover, the sustained actin polymerization phase observed in native rat MTAL cells was absent in cultured HL60 cells (19). These different patterns of actin cytoskeleton remodeling are associated with differences in RVI efficacy. Indeed, in contrast to the majority of cells exhibiting little or no RVI, MTAL cells undergo robust RVI (35, 43).

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 actin polymerization resulting in the generation of a diffuse network of F-actin bundles play an important role in the RVI of MTAL cells. Results of the present study indicate that whole cell actin depolymerization with latrunculin B facilitates RVI, whereas global inhibition of actin depolymerization by jasplakinolide antagonizes RVI (see Figs. 3 and 4). These results suggest that depolymerization of F-actin is required for RVI in MTAL epithelial cells. In addition, fluorescence microscopy imaging shows that RVI is associated with reduced cortical F-actin staining (see Fig. 2), suggesting that the F-actin depolymerization process involved in RVI specifically takes place at the level of the cortical F-actin ring in MTAL epithelial cells. This result contrasts with those obtained in nonepithelial cells (HL60) that exhibit a densification of the cortical F-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 and reduced the efficacy of RVI (see Figs. 4 and 6). These results suggest that, in addition to cortical F-actin ring depolymerization, the generation of a dense diffuse network of F-actin bundles facilitates RVI. At fist glance, this finding contrasts with the effect of jasplakinolide, which increases the actin polymerization level and prevents the RVI. It should be noticed, however, that jasplakinolide also increased actin polymerization at the level of the cortical 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/HCO3 exchanger, and Na-K-ATPase (4, 15, 29, 43, 47), which might, at least in part, be dependent on cortical actin polymerization level. For instance, inhibition of F-actin depolymerization by phalloidin or jasplakinolide impairs the 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 may control the activity of ion transporters in different ways. A shift in F-actin-G-actin equilibrium toward G-actin may stimulate the activity of specific 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 of ion, solute, and water transporters as demonstrated for the Na/H exchanger NHE3 (11), volume-sensitive Cl channels (33), the glucose transporter GLUT4 (26), and the water channel aquaporin-2 (22). The results 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-actin bundles (see Fig. 2) that may play 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 increased the extent of maximal cell shrinkage and reduced the efficacy of RVI in response to hypertonicity (see Figs. 4, 6, and 7). This effect might be partly achieved through mechanical constraints exerted on the cell membrane, as described for lamellipodia or filipodia formation (44). It occurs, however, most likely indirectly via spacial control of signaling events and/or facilitation of the delivery of ion transporters from intracellular stores to the plasma membrane, 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 the actin cytoskeleton may be part of the osmosensing machinery was considered. Our results, however, suggest that actin cytoskeleton remodeling and integrity are not essential for the activation of MAPKs in response to increased extracellular osmolality. Indeed, interfering with neither the polymerization level 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 cytoplasmic concentration of macromolecules (34) or aggregation of growth factor and cytokine receptors leading to their ligand-independent activation (36) have to be considered.

In conclusion, we showed that an acute extracellular hypertonic challenge induces actin cytoskeleton remodeling consisting of F-actin redistribution from a cortical ring to a diffuse network of F-actin bundles in native rat MTAL cells. We propose the following working hypothesis summarized by Fig. 8. Cell shrinkage induces p38 kinase activation, which in turn, promotes cortical F-actin ring depolymerization and generation of a dense diffuse network of F-actin bundles that both promote RVI most likely through modulation of ion transporter activity. Further investigation is required to identify the molecular players involved in actin cytoskeleton remodeling. In addition, the role of actin cytoskeleton remodeling in the control of the activity and/or abundance of ion transporters involved in RVI remains to be determined.



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Fig. 8. Schematic representation of the sequence of events linking p38 kinase activation, actin cytoskeleton, and regulatory volume increase (RVI) in MTAL epithelial cells. Exposure of MTAL epithelial cells to extracellular hypertonicity induces cell shrinkage leading to the activation of p38 kinase. Increased p38 kinase activity promotes depolymerization of the cortical F-actin ring and polymerization of new actin filaments generating a dense diffuse network of F-actin bundles. Both processes may stimulate the activity of ion transporters leading to intracellular ion accumulation, secondary water influx, and recovery of the initial cellular volume.

 


    DISCLOSURES
 
This work was supported in part by Grants 31–50–643.97 and 31–56830.99 from the Swiss National Foundation to E. Féraille and by a grant from the Fondation Novartis pour la Recherche en Sciences Médico-biologiques to E. Féraille.


    ACKNOWLEDGMENTS
 
We thank Dr. C. Chaponnier for helpful discussions and critical reading of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. Féraille, Division de Néphrologie, Fondation pour Recherches Médicales, CH-1211 Genève 4, Switzerland (E-mail: Eric.Feraille{at}medecine.unige.ch).

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.


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