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Abstract In the renal medullary thick ascending limb (MTAL), inhibiting the basolateral NHE1 Na +/H + exchanger with amiloride or nerve growth factor (NGF) results secondarily in inhibition of the apical NHE3 Na +/H + exchanger, thereby decreasing transepithelial absorption. MTALs from rats were studied by in vitro microperfusion to identify the mechanism underlying cross-talk between the two exchangers. The basolateral addition of 10 μ m amiloride or 0.7 n m NGF decreased absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on absorption in tubules bathed with amiloride or a Na +-free bath to inhibit NHE1.
Jasplakinolide and latrunculin B did not prevent inhibition of absorption by vasopressin or stimulation by hyposmolality, factors that regulate absorption through primary effects on apical Na +/H + exchange. Treatment of MTALs with amiloride or NGF for 15 min decreased polymerized actin with no change in total cell actin, as assessed both by fluorescence microscopy and by actin Triton X-100 solubility. Jasplakinolide prevented amiloride-induced actin remodeling.
Vasopressin, which inhibits absorption by an amount similar to that observed with amiloride and NGF but does not act via NHE1, did not affect cellular F-actin content. These results indicate that basolateral NHE1 regulates apical NHE3 and absorption in the MTAL by controlling the organization of the actin cytoskeleton. Na +/H + exchangers (NHEs) are transmembrane proteins that mediate the electroneutral exchange of Na + for H + (, ). At least eight NHE isoforms (NHE1 to -8) have been identified in mammalian cells (-). These differ in their tissue distribution, membrane localization, inhibitor sensitivity, and physiological functions. NHE1 is expressed ubiquitously in the plasma membrane of nonpolarized cells and in the basolateral membrane of epithelial cells, where it mediates housekeeping functions such as regulation of intracellular pH and cell volume (-, ). Activation of NHE1 also is involved in other important cellular processes, including growth, migration, survival, and adhesion (, ).
In contrast, other exchangers (NHE2 to -5) exhibit a more restricted tissue distribution (, ). In particular, NHE3 is localized selectively to the apical membrane of epithelial cells in the kidney and gastrointestinal tract, where it mediates the transepithelial absorption of NaCl and/or NaHCO 3 (, -). Regulation of NHE3 activity in the kidney is important for the maintenance of acid-base balance, Na + balance and extracellular fluid volume, and blood pressure. The medullary thick ascending limb (MTAL) of the mammalian kidney participates in acid-base homeostasis by reabsorbing most of the filtered not reabsorbed by the proximal tubule (, ). Absorption in the MTAL depends on luminal H + secretion mediated by the apical membrane NHE3 Na +/H + exchanger (, -).
The MTAL also expresses basolateral NHE1 (-), and we have recently identified a novel role for this exchanger in transepithelial absorption. Inhibition of basolateral Na +/H + exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na +/H + exchange, thereby decreasing absorption (, ). NHE1 was identified conclusively as the basolateral exchanger responsible for this regulation based on the observations that basal absorption is reduced, and inhibition of absorption by basolateral amiloride and NGF is eliminated, in MTALs from NHE1 null mutant mice. The control of absorption in the MTAL thus involves cross-talk between the basolateral and apical membrane Na +/H + exchangers, whereby basolateral NHE1 enhances the activity of apical NHE3 (-). The effect of NHE1 to increase apical NHE3 activity cannot be explained by a change in the net driving force for the apical exchanger and thus appears to be mediated through a signal transduction pathway (, ). However, the identity of this signaling pathway has remained undefined.
The cytoskeleton plays a role in the targeting, anchoring, and regulation of a variety of ion transport proteins. Recent work has identified important regulatory interactions between the actin cytoskeleton and NHEs. NHE1 binds to actin filaments and anchors the actin cytoskeleton to the plasma membrane (, ).
NHE1 is also a component of signaling pathways that regulate several cytoskeleton-dependent processes, including cell adhesion and motility, and selective inhibition of NHE1 has been shown to impair actin filament assembly in cell lines (, -). Thus, NHE1 is involved in regulating the assembly and organization of the actin cytoskeletal network. The epithelial NHE3 Na +/H + exchanger is linked indirectly to the cytoskeleton via actin-binding adapter proteins such as ezrin (, ), and cytoskeletal interactions play a role in the regulation of NHE3 by factors such as cAMP and endothelin (-). In an antiporter-deficient fibroblast cell line transfected with NHE3, either stabilizing or depolymerizing actin filaments markedly inhibited NHE3 by decreasing its intrinsic activity. When considered in the context of epithelial cells such as the MTAL that express NHE1 in the basolateral membrane and NHE3 in the apical membrane, the above findings raise the possibility that the cytoskeleton could mediate cross-talk between the two exchangers, whereby NHE1 could induce changes in the actin network that in turn modulate NHE3. The purpose of the present experiments was to investigate the role of the actin cytoskeleton in transepithelial absorption in the MTAL and to determine whether the cytoskeleton mediates the regulatory interaction between the basolateral NHE1 and apical NHE3 Na +/H + exchangers. The results demonstrate that NHE1 regulates the organization of the actin cytoskeleton in the MTAL and that actin remodeling is involved in mediating NHE1-induced regulation of apical NHE3 and absorption.
EXPERIMENTAL PROCEDURES Tubule Perfusion and Measurement of Net Absorption—MTALs from male Sprague-Dawley rats (50-100-g body weight; Taconic, Germantown, NY) were isolated and perfused in vitro as described (, ). Tubules were dissected from the inner stripe of the outer medulla at 10 °C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37 °C. In most experiments, the tubules were perfused and bathed in control solution that contained 146 m m Na +, 4 m m K +, 122 m m Cl -, 25 m m, 2.0 m m Ca 2+, 1.5 m m Mg 2+, 2.0 m m phosphate, 1.2 m m, 1.0 m m citrate, 2.0 m m lactate, and 5.5 m m glucose (osmolality = 295 mosmol/kg H 2O). In one series of experiments , Na + in the bath solution was replaced completely with N-methyl- d-glucammonium (, ). Hyposmotic solutions (245 mosmol/kg H 2O) (Figs.
And ) were produced by removing 25 m m NaCl from the control solution or by removing 50 m m mannitol from control solution in which 50 m m mannitol replaced 25 m m NaCl. Bath solutions contained 0.2 g/100 ml fatty acid-free bovine albumin. All solutions were equilibrated with 95% O 2, 5% CO 2 and were pH 7.45 at 37 °C. Experimental agents were added to the bath solutions as described under “Results.” Jasplakinolide and latrunculin B were prepared as stock solutions in dimethyl sulfoxide and diluted into bath solutions to final concentrations given under “Results.” Equal concentrations of vehicle were added to control solutions. Solutions containing other experimental agents were prepared as described (, ). Conditions that inhibit basolateral Na +/H + exchange prevent inhibition of absorption by jasplakinolide.
MTALs were studied with 10 μ m amiloride in the bath ( A) or in a Na +-free bath ( B), and then 0.05 μ m jasplakinolide was added to the bath. B, Na + in the bath solution was replaced completely with N-methyl- d-glucammonium; the lumen was perfused with control solution containing 146 m m Na + (, ) (see “Experimental Procedures”)., data points, lines, and p values are as in.
NS, not significant. Mean values are given under “Results.”. Jasplakinolide does not prevent regulation by vasopressin (AVP) and hyposmolality. MTALs were studied in control solution with 0.05 μ m jasplakinolide ( Jasp) in the bath for 25-50 min, and then 10 -10 m AVP was added to the bath ( A), or hyposmolality was produced in the lumen and bath ( B). In B, the osmolality was decreased from 295 mosmol/kg H 2O to 245 mosmol/kg H 2O ( Hypo) by removing either 50 m m mannitol ( filled circles) or 25 m m NaCl ( open circles) from the lumen and bath solutions., data points, lines, and p values are as in. Mean values are given under “Results.”. The protocol for study of transepithelial absorption was as described (, ).
In most experiments, tubules were equilibrated for 20-30 min at 37 °C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit of tubule length) was adjusted to 1.6-2.0 nl/min/mm. 1-5 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5-10 min after an experimental agent was added to or removed from the bath solution. In some experiments, longer treatment periods were used, as described under “Results.” The absolute rate of absorption (, pmol/min/mm) was calculated from the luminal flow rate and the difference between total CO 2 concentrations measured in perfused and collected fluids.
An average absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in the figures. ( n = number of tubules) are presented under “Results”.
Phalloidin Staining and Fluorescence Microscopy—MTALs were microdissected and mounted on Cell-Tak-coated coverslips at 4 °C. The tubules were then incubated in a flowing bath at 37 °C using the same solutions and protocols as in transport experiments (see “Results”). Following incubation, the tubules were washed with phosphate-buffered saline (PBS) and fixed for 15 min at room temperature with 4% paraformaldehyde in PBS. After washing in PBS, the tubules were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. The tubules were then blocked with PBS plus 1% bovine serum albumin for 20 min at room temperature, washed, and incubated with Alexa 488-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR) in blocking solution (1:200) for 1 h at room temperature to label F-actin. After staining, the tubules were washed and placed in fresh PBS for fluorescence imaging. Fluorescence staining was examined using a Zeiss confocal microscope (LSM 510 META) equipped with a ×63 oil immersion objective.
The tubules were imaged longitudinally, and optical sections were obtained with a step displacement of. RESULTS Bath Amiloride and NGF Inhibit Absorption—Adding 10 μ m amiloride to the bath decreased absorption in isolated rat MTALs by 27%, from 14.2 ± 0.7 to 10.4 ± 0.8 pmol/min/mm ( p. Effects of Jasplakinolide on Absorption—To investigate the role of the cytoskeleton in absorption, we examined the effects of jasplakinolide, a membrane-permeant cyclic peptide that binds and stabilizes actin filaments and promotes actin polymerization. Adding 0.05 μ m jasplakinolide to the bath decreased absorption by 32%, from 14.8 ± 0.3 to 10.0 ± 0.4 pmol/min/mm ( p.
Jasplakinolide ( Jasp) blocks inhibition by bath amiloride and NGF. A, MTALs were studied in control solution, and then 0.05 μ m jasplakinolide was added to and removed from the bath solution. B and C, tubules were bathed with 0.05 μ m jasplakinolide for 25-30 min, and then 10 μ m amiloride ( B) or 0.7 n m NGF ( C) was added to the bath solution. Tubules were exposed to amiloride or NGF for at least 20 min., data points, lines, and p values are as in. NS, not significant. Mean values are given under “Results.”. To assess the specificity of jasplakinolide's actions on the regulation of absorption, we examined the effects of vasopressin (AVP) and hyposmolality.
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These factors were studied because AVP inhibits and hyposmolality stimulates absorption in the MTAL through primary effects on the apical Na +/H + exchanger (, ), contrary to bath amiloride and NGF, which act primarily on the basolateral Na +/H + exchanger. In MTAL bathed with 0.05 μ m jasplakinolide for 25-50 min, adding 10 -10 m AVP to the bath decreased absorption by 43%, from 11.4 ± 0.5 to 6.5 ± 0.9 pmol/min/mm ( p.
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As with jasplakinolide, the specificity of latrunculin B action was tested using AVP and hyposmolality. In MTAL bathed with 1 μ m latrunculin B for 100 min, AVP decreased absorption from 9.3 ± 0.7 to 4.2 ± 0.3 pmol/min/mm ( p. Effects of Inhibitors of NHE1 on the Actin Cytoskeleton—To examine more directly possible interactions between NHE1 and the cytoskeleton, F-actin was studied in microdissected MTALs by Alexa 448-phalloidin staining and confocal fluorescence microscopy. Tubules were optically sectioned, three-dimensional images were constructed, and the fluorescence intensity of phalloidin staining normalized for tubule volume was quantified.
MTALs exhibit F-actin staining along the inner surface of the plasma membranes (cortical actin), a diffuse F-actin network throughout the cytoplasm, and a dense annular bundle of actin filaments surrounding the apical cell pole (actin belt of the zonula adherens). Treatment with either 10 μ m amiloride or 0.7 n m NGF for 15 min decreased the intensity of fluorescence staining by 30% ( p.
Amiloride and NGF induce actin cytoskeleton reorganization in the MTAL. A, microdissected MTALs were incubated in vitro in control solution ( Cont), 10 μ m amiloride ( Amil), 0.7 n m NGF, or 10 -10 m AVP for 15 min or with 1 μ m latrunculin B ( Lat B) for 60 min. Results for 60-min controls (not shown) did not differ from 15-min controls. Tubules were fixed, permeabilized, and stained with Alexa 488-phalloidin to visualize F-actin. Three-dimensional images were constructed from longitudinal optical sections. Treatment with 1 μ m latrunculin B for 60 min decreased fluorescence intensity , consistent with its action to depolymerize F-actin.
Latrunculin B disrupted the cortical and cytoplasmic F-actin networks, with relative preservation of the annular F-actin bundle. Latrunculin B had no effect on fluorescence intensity at 15 min (not shown). Thus, the time course for latrunculin B-induced disruption of actin filaments parallels its effects on absorption. Effects of Inhibitors of NHE1 on Actin Solubility—To examine further the interaction of NHE1 with the cytoskeleton, the relative amounts of actin in Triton X-100-soluble and -insoluble fractions were determined. Because actin filaments are resistant to mild detergent extraction, quantification of the fraction of Triton X-100-insoluble actin provides a measure of cellular F-actin content and the extent of actin polymerization (, ). Inner stripe tissue was incubated in vitro using the same solutions and treatments as in transport and fluorescence imaging experiments.
In tissue incubated in control solution, 40% of cellular actin is in the soluble form, and 60% is in the insoluble (F-actin) form. This distribution did not differ for control incubations of 15 or 60 min. Treatment with either 10 μ m amiloride or 0.7 n m NGF for 15 min reversed the distribution, increasing soluble actin from 40 to 60% and decreasing insoluble actin from 60 to 40% ( p. Effect of NHE1 inhibitors and latrunculin B on actin solubility. A, inner stripe tissue was incubated in vitro in control solution ( Cont), 10 μ m amiloride, or 0.7 n m NGF for 15 min or with 1 μ m latrunculin B ( Lat B) for 60 min. Results for 60-min controls (not shown) did not differ from 15-min controls.
Tissue samples were extracted with 1% Triton X-100, and actin in soluble and insoluble fractions was analyzed by immunoblotting. Actin immunoblots are for representative experiments. B, immunoreactive bands were quantified by densitometry, and actin in Triton X-100-soluble and -insoluble fractions was expressed as the percentage of total actin. Data are means ± S.E. For 4-8 experiments in each condition. Further experiments examining actin detergent solubility were carried out to verify that the effect of jasplakinolide to block NHE1-mediated regulation of absorption was related to an effect on the cytoskeleton.
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This approach was used because effects of jasplakinolide on F-actin content cannot be quantified by phalloidin labeling due to the fact that jasplakinolide binds to F-actin competitively with phalloidin. Inner stripe tissue was incubated in control solution in the absence and presence of 0.05 μ m jasplakinolide for 15 min. The tissue was then either maintained in these solutions or treated with 10 μ m amiloride for an additional 15 min.
Treatment with jasplakinolide alone did not alter actin detergent solubility. However, jasplakinolide blocked completely the effects of amiloride to increase the Triton X-100-soluble fraction and to decrease the Triton X-100-insoluble fraction of cellular actin. Thus, in the MTAL, jasplakinolide blocks both the effects of basolateral amiloride to induce F-actin remodeling and to inhibit absorption.
Jasplakinolide blocks amiloride-induced actin reorganization. A, inner stripe tissue was incubated in vitro in control solution in the absence and presence of 0.05 μ m jasplakinolide for 15 min. The tissue was then either maintained in these solutions or treated with 10 μ m amiloride for an additional 15 min. Actin in Triton X-100-soluble and -insoluble fractions was analyzed by immunoblotting. Immunoblots are for a representative experiment.
B, immunoreactive bands were quantified by densitometry, and actin in soluble and insoluble fractions was expressed as the percentage of total actin. Data are means ± S.E.
For four experiments. DISCUSSION Previously, we identified an important role for the basolateral NHE1 Na +/H + exchanger in regulation of transepithelial absorption in the MTAL. This involves a novel and paradoxical mechanism whereby inhibition of basolateral NHE1 results secondarily in the inhibition of apical NHE3, thereby decreasing absorption (-). These results provided the first evidence of a regulatory role for NHE1 in transepithelial transport in the kidney. In the present study, we examined the mechanism of cross-talk between the two Na +/H + exchangers.
The results show that NHE1 regulates NHE3 and absorption by controlling the organization of the actin cytoskeleton. Our data support a model in which inhibition of basolateral NHE1 induces a decrease in polymerized actin that inhibits the apical NHE3 Na +/H + exchanger. Basolateral NHE1 regulates apical NHE3 and absorption through cytoskeleton remodeling. Transepithelial absorption in the MTAL is mediated by H + secretion via apical NHE3 and efflux of across the basolateral membrane via mechanisms not yet established.
Inhibiting NHE1 with amiloride or NGF induces reorganization of F-actin that decreases NHE3 activity and absorption. The cell model is modified from Ref. ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. The conclusion that NHE1 regulates NHE3 via the cytoskeleton is supported by functional and biochemical evidence.
Footnotes. 1 The abbreviations used are: NHE, Na +/H + exchanger (the number following “NHE” refers to the specific isoform); MTAL, medullary thick ascending limb; NGF, nerve growth factor; AVP, arginine vasopressin; PBS, phosphate-buffered saline.
2 Jasplakinolide has been shown to increase the fraction of Triton X-100-insoluble (polymerized) actin in some systems (, ). However, these studies used much higher jasplakinolide concentrations (1-10 μM) and longer exposure times (45-120 min) than those used in our experiments (0.05 μ m for 15-30 min). We used a relatively low dose of jasplakinolide that is above the IC 50 for F-actin binding , induced a rapid, stable, and reversible inhibition of absorption, and blocked amiloride-induced F-actin rearrangement. The stabilizing effect of jasplakinolide on F-actin in our experiments did not result in a measurable change in actin Triton X solubility. This work was supported by National Institutes of Health Grant DK-38217 and by a grant from the John Sealy Memorial Endowment Fund for Biomedical Research. The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “ advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 17, 2004. Revision received December 20, 2004. The American Society for Biochemistry and Molecular Biology, Inc.