Endothelial nitric oxide synthase (eNOS) regulates NaCl absorption by the thick ascending limb of the loop of Henle (THAL). We found that augmenting luminal flow induces eNOS activation and translocation to the apical membrane of THALs (Ortiz PA, Hong NJ, and Garvin JL. Am J Physiol Renal Physiol 287: F274–F280, 2004). In other cells, eNOS activation by shear stress is mediated by phosphatidylinositol 3-OH kinase (PI3)-kinase. We hypothesized that luminal flow induces eNOS activation via PI3-kinase. Pretreatment of THALs with wortmannin, a PI3-kinase inhibitor, significantly reduced flow-induced nitric oxide (NO) release by 75% (from 53.6 ± 6 to 13.2 ± 5.7 pA/mm). Increasing luminal flow from 0 to 20 nl/min induced eNOS translocation to the apical membrane, whereas in the presence of wortmannin eNOS translocation was prevented (basolateral = 32 ± 2%, middle = 38 ± 1%, apical = 30 ± 1%, n = 5, not significant vs. no flow). We next studied which PI3-kinase product mediates eNOS translocation. Addition of PI(3,4,5)P3 (5 μM) in the absence of flow increased NO levels (P < 0.05) and induced eNOS translocation to the apical membrane (from 40 ± 4 to 60 ± 2% of total eNOS, n = 6, P < 0.05). Incubation with PI(3,4)P2 or PI(4,5)P2 did not change eNOS localization. We next tested whether heat shock protein (Hsp)90 is involved in eNOS translocation. The Hsp90 inhibitor geldanamycin blocked flow-induced eNOS translocation to the apical membrane (n = 6). Flow also induced translocation of Hsp90 to the apical membrane (from 35 ± 2 to 57 ± 2%; P < 0.05) in a PI3-kinase-dependent manner. We conclude that luminal flow induces eNOS translocation and activation in the THAL via PI3-kinase and that Hsp90 is involved in eNOS translocation to the apical membrane.
- nitric oxide
- renal tubules
- heat shock protein
- endothelial nitric oxide synthase
in polarized epithelial cells of the kidney, respiratory tract, and testis, nitric oxide (NO) produced by endothelial NO synthase (eNOS) regulates important cellular functions (14, 27, 29, 34, 55, 57, 60). Thus eNOS activity and NO production must be tightly regulated in these cells. We previously reported that NO acts as an autacoid to inhibit NaCl and bicarbonate absorption by the thick ascending limb of the loop of Henle (THAL) (31, 32, 39). More recently, we identified eNOS as the NOS isoform responsible for the regulation of NaCl absorption by the THAL (35, 38). We recently observed that increasing luminal flow acutely stimulated eNOS activity and induced translocation of eNOS from the basolateral membrane and cytoplasm to the apical membrane of isolated THALs (34a). However, the signaling cascade that mediates eNOS activation and translocation by flow is unknown.
Regulation of eNOS has been studied in depth in vascular endothelial cells, where one of the most potent activators of eNOS is flow-induced shear stress (5, 46). The mechanism by which flow activates eNOS in these cells is independent of changes in intracellular calcium and involves activation of the phosphatidylinositol 3-OH kinase (PI3-kinase) cascade (11). Moreover, increased flow induces phosphorylation and activation of eNOS by the serine/threonine kinase Akt in a manner dependent on intact PI3-kinase activity (9, 16). Some eNOS agonists that act via PI3-kinase, such as vascular endothelial growth factor (VEGF) and estradiol, not only increase enzymatic activity but also induce changes in eNOS subcellular localization (19, 40). We hypothesized that PI3-kinase mediates flow-induced eNOS activation and translocation in the THAL. We studied eNOS localization and NO production in freshly isolated THALs and found that luminal flow induced eNOS activation and translocation to the apical membrane in a PI3-kinase-dependent manner. The PI3-kinase product PI(3, 4,5)P3 induced eNOS translocation and activation in the absence of luminal flow, whereas another PI3-kinase product, PI(3,4)P2, did not. Increasing luminal flow also induced translocation of heat shock protein (Hsp)90, a chaperone protein known to interact with eNOS, to the apical membrane of THALs. We concluded that PI3-kinase and its specific product PI(3,4,5)P3 mediate flow-induced eNOS translocation and activation in the THAL and that Hsp90 is required for eNOS translocation.
Dissection and perfusion of THALs.
Male Sprague-Dawley rats weighing 120–150 g (Charles River Breeding Laboratories, Wilmington, MA) were fed a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) for at least 5 days. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). After anesthesia, the abdominal cavity was opened, and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. THALs under a stereomicroscope were dissected from the medullary rays at 4–10°C, transferred to a temperature-regulated chamber, and held between concentric glass pipettes at 37 ± 1°C as performed routinely in our laboratory. All drugs were added to the bath before the experiment. Phosphatidylinositol(3,4,5)P3-C16, phosphatidylinositol(4,5)P2-C16, and phosphatidylinositol(3,4)P2-C16 (Echelon Biosciences, Salt Lake City, UT) were freshly prepared before the experiments and mixed with the intracellular carrier neomycin B sulfate at equimolar concentrations 20 min before addition to the bath for a final concentration of 5 μM, according to the vendor's protocol and published material (36, 53).
All protocols involving animals have been approved by the Institutional Animal Care and Use Committee at Henry Ford Hospital.
Immunodetection of eNOS and Hsp90 in isolated THALs using confocal microscopy.
Tubules were fixed for 15 min with 4% paraformaldehyde in 150 mM NaCl and 10 mM Na2HPO4, pH 7.4. Fixed cells were blocked for 30 min with 1% BSA in TBS-T perfused into the lumen followed by a 30-min incubation with primary antibodies diluted in 1% BSA in TBS-T (eNOS, Hsp90, 1:1,000) in the lumen. Cells were washed with TBS-T for 5 min in both lumen and bath and then incubated for 30 min with a secondary antibody conjugated to a fluorescent dye (Alexa Fluor 488 goat anti-mouse IgG) diluted 1:200 in 1% BSA in TBS-T. Cells were then washed for 5 min with TBS-T in lumen and bath, and fluorescent images were acquired using a confocal microscope as described in detail elsewhere (34a).
Monoclonal antibodies to eNOS were obtained from BD Transduction Labs (San Diego, CA); Anti-Hsp90 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488 goat anti-mouse IgG was from Molecular Probes (Eugene, OR). Cytochalasin D and wortmannin were purchased from Sigma (St. Louis, MO).
Microelectrode NO measurements.
NO released by THALs was measured using an amperometric microelectrode selective for NO (inNO measuring system, Harvard Apparatus) as described in detail elsewhere (33, 34a). After a 30-min equilibration period in which a stable baseline was achieved (±5 pA), NO production was measured in the absence of luminal flow for 10 min and averaged as basal NO production. Then, flow was increased to 20–25 nl/min and NO release was monitored continuously until it reached a new level, at which point a new 10-min period was measured and averaged. The difference between baseline (0 nl/min) and the level reached after luminal flow was increased (20–25 nl/min) was taken as the response and normalized per tubule length (mm) as described previously (33). When the effect of wortmannin on NO production was tested, this was added to the bath at the beginning of the experiment.
NO measurement with diaminofluorescein-2 diacetate.
The effect of phosphatidylinositol(3,4,5)P3 on NO production in isolated THALs in the absence of luminal flow was measured by monitoring the changes in intracellular fluorescence of the NO-sensitive dye diaminofluorescein-2 diacetate (DAF-2DA; Calbiochem, La Jolla, CA) (30). Briefly, THALs were loaded with DAF-2DA present in the bath (final concentration 4 μmol/l) for 35 min and then washed in perfusion solution for 20 min. The dye was excited with an argon laser set at 488 nm, and the fluorescence emitted by NO-bound dye was measured using a laser-scanning confocal microscope (Noran Instruments, Middleton, WI). Measurements were recorded once every minute for a 5-min control period. Then, phosphatidylinositol(3,4,5)P3 (5 μM final concentration) was added to the bath, and 10 min later fluorescence was measured during a 10-min experimental period. The carrier for intracellular delivery of phosphoinositides, neomycin B sulfate, had no effect on baseline fluorescence.
Results are expressed as means ± SE. Student's paired t-test was used to determine statistical differences between means before and after treatment in the same group of tubules. One-way ANOVA was used to determine statistical differences between different groups. P < 0.05 was considered significant.
We recently observed (34a) that increasing luminal flow acutely stimulated eNOS activity and induced translocation of eNOS to the apical membrane of THALs. In this study, we investigated the signaling cascade mediating these events. We first tested whether blockade of PI3-kinase could prevent flow-induced NO production by THALs. Under control conditions, increasing luminal flow stimulated NO release by 53.6 ± 6.0 pA/mm of tubule length (n = 5, P < 0.05). In a different set of tubules preincubated with the PI3-kinase inhibitor wortmannin (150 nM), increasing luminal flow only stimulated NO release by 13.2 ± 5.7 pA/mm of tubule, a 75% blockade (n = 5, P < 0.01) (Fig. 1). These data suggest that PI3-kinase is part of the cascade that mediates eNOS activation by luminal flow in the THAL.
Because PI3-kinase was found to be involved in eNOS activation, we next tested whether blockade of PI3-kinase could also prevent flow-induced eNOS translocation to the apical membrane of THALs. In the absence of flow, eNOS was diffusely distributed throughout the cell cytoplasm and apical and basolateral membranes (basolateral membrane = 33 ± 3%, middle = 27 ± 2%, apical membrane = 40 ± 2% of total fluorescence intensity) (Fig. 2), whereas in the presence of luminal flow (20–25 nl/min) eNOS was translocated to the apical membrane (from 40 ± 2 to 64 ± 2%, n = 6; P < 0.05). Preincubation of THALs with wortmannin (150 nM) completely blocked flow-induced eNOS translocation to the apical membrane [basolateral membrane = 32 ± 3%, middle = 38 ± 1%, apical membrane = 30 ± 1%, n = 5; not significant (NS) vs. no flow] (Fig. 2). In control experiments, wortmannin (150 nM) did not significantly affect eNOS localization in the absence of luminal flow (basolateral membrane = 33 ± 1%, middle = 32 ± 1%, apical membrane = 35 ± 1%, n = 3). These data suggest that activation of PI3-kinase mediates the effects of flow on eNOS translocation to the apical membrane of THALs.
Activation of PI3-kinase induces the formation of PI(3,4,5)P3. Thus we tested whether the addition of exogenous PI(3,4,5)P3 activates eNOS in the absence of luminal flow. THALs were held between glass pipettes and left unperfused. Intracellular NO levels were monitored by measuring changes in fluorescence of the NO-sensitive dye DAF-2DA. We found that during the control period, intracellular fluorescence averaged 67.5 ± 10.7 arbitrary fluorescence units (AU). Twenty minutes after addition of PI(3,4,5)P3 (5 μM) to the bath, intracellular fluorescence increased to 75.3 ± 12.0 AU, an average 11.3 ± 1.8% increase (n = 6; P < 0.01) (data not shown). Control experiments showed no change in fluorescence over time (from 57.7 ± 4.9 to 55.7 ± 3.8 AU, n = 3) or with the addition of the carrier alone. These data indicate that PI(3,4,5)P3 stimulates eNOS activity in the THAL in the absence of luminal flow.
Because PI(3,4,5)P3 induced eNOS activation, we next tested whether the addition of PI(3,4,5)P3 could induce eNOS translocation to the apical membrane in the absence of luminal flow. We found that 30 min after incubation with PI(3,4,5)P3 (5 μM) using neomycin B sulfate for intracellular delivery, eNOS was translocated to the apical membrane (from 40 ± 2 to 60 ± 1%, n = 6; P < 0.05) (Fig. 3). In contrast, after incubation with PI(4,5)P2 (5 μM) or PI(3,4)P2 (5 μM) eNOS was diffusely distributed throughout the cell, similar to the absence of luminal flow [no flow+PI(4,5)P2: apical membrane = 35 ± 1%, n = 4; no flow+PI(3,4)P2: apical membrane = 37 ± 1%, n = 4] (Fig. 3). Taken together, these data indicate that the PI3-kinase product PI(3,4,5)P3 induces eNOS translocation and activation in the absence of luminal flow, pointing out the importance of the PI3-kinase cascade and this specific product in regulating eNOS subcellular localization and activity.
The chaperone protein Hsp90 directly binds eNOS and is also involved in PI3-kinase-dependent eNOS activation (13, 17, 44). Thus we tested whether Hsp90 is involved in eNOS translocation in the THAL using geldanamycin, an antibiotic that inhibits Hsp90 ATPase activity (4, 47). We found that preincubation of THALs with geldanamycin (1 μM) completely blocked flow-induced eNOS translocation to the apical membrane (no flow = 40 ± 2%; flow = 64 ± 2%; flow+geldanamycin = 36 ± 1%, n = 6) (Fig. 4). In control experiments, geldanamycin (1 μM) did not significantly affect eNOS localization in the absence of luminal flow (basolateral membrane = 34 ± 2%, middle = 29 ± 2%, apical membrane = 37 ± 2%, NS vs. no flow, n = 2). These data suggest that Hsp90 is necessary for flow to induce eNOS translocation to the apical membrane of THALs.
Because geldanamycin completely blocked flow-induced eNOS translocation to the apical membrane, we tested whether flow induces Hsp90 translocation to the apical membrane. We found that in the absence of flow, Hsp90 was diffusely distributed throughout the cell cytoplasm and apical and basolateral membranes. Thirty minutes after luminal flow was increased, we observed a significant increase in immunoreactive Hsp90 at the apical membrane (from 35 ± 2 to 57 ± 2%; n = 6, P < 0.05), whereas the amount of Hsp90 in the basolateral membrane and cytoplasm decreased from 30 ± 3 to 17 ± 1 and from 35 ± 2 to 25 ± 2%, respectively (n = 6; P < 0.05 vs. no flow) (Fig. 5). Taken together, these data indicate that intact Hsp90 activity is required for eNOS translocation and that Hsp90 itself traffics to the apical membrane when stimulated by flow.
Finally, we tested whether Hsp90 translocation was dependent on PI3-kinase activation by flow. We found that preincubation of THALs with wortmannin (150 nM) completely blocked flow-induced Hsp90 translocation to the apical membrane (basolateral membrane = 33 ± 2%, middle = 35 ± 2%, apical membrane = 32 ± 2%, n = 4; NS vs. no flow) (Fig. 5). These data suggest that Hsp90 translocation is dependent on activation of PI3-kinase by flow.
We found that increasing luminal flow induced eNOS activation and eNOS translocation to the apical membrane of THALs. Blockade of PI3-kinase with wortmannin prevented flow-induced eNOS activation and translocation. The addition of the PI3-kinase product PI(3,4,5)P3 induced eNOS translocation in the absence of luminal flow and also increased intracellular NO levels. In contrast, PI(3,4)P2 or PI(4,5)P2 did not induce eNOS translocation. Finally, we found that a Hsp90 inhibitor blocked flow-induced eNOS translocation, and flow caused the translocation of Hsp90 itself to the apical membrane. We concluded that flow induces eNOS translocation and activation via activation of the PI3-kinase cascade and formation of the specific product PI(3,4,5)P3. Hsp90 is necessary for eNOS translocation to the apical membrane in response to increased luminal flow. Given the importance of eNOS-derived NO in the regulation of THAL NaCl absorption (35, 38) and the role of eNOS in various physiological processes in epithelial cells of the respiratory tract, brain, and testis (14, 29, 55, 57, 60), this mechanism is likely to play an essential role in the regulation of NO levels in these cells.
We found that increasing luminal flow from 0 to 20–25 nl/min stimulated eNOS activity and induced eNOS translocation to the apical membrane of THALs (34a). In endothelial cells, flow-induced shear stress activates eNOS via PI3-kinase, which results in eNOS phosphorylation at serine 1179 by Akt (11, 15, 16). We examined the role of the PI3-kinase cascade in flow-induced eNOS activation and translocation. We found that the PI3-kinase inhibitor wortmannin completely blocked flow-induced eNOS translocation and blunted flow-induced NO production. The PI3-kinase product PI(3,4,5)P3, but not PI(3,4)P2 or the precursor PI(4,5)P2, caused eNOS translocation to the apical membrane and increased eNOS activity in the absence of luminal flow. These data indicate that the PI3-kinase cascade mediates flow-induced eNOS translocation and activation in the THAL and that activation of this signaling pathway is sufficient to induce eNOS translocation and activation. PI3-kinase inhibition blocked eNOS activation and translocation; thus it is likely that the same signaling cascade that causes eNOS activation also induces eNOS translocation. However, it is currently not clear whether eNOS activation and translocation depend solely on Akt, because PI3-kinase may also regulate protein trafficking independently of Akt activation.
eNOS activation by estradiol and VEGF in endothelial cells is dependent on PI3-kinase activity (18, 25). In addition, these two agonists also induce rapid eNOS translocation from the plasma membrane to the cytoplasm of cultured endothelial cells (10, 19). However, it was not known whether PI3-kinase was involved in eNOS translocation in these cells. We believe our data show for the first time that PI3-kinase and its specific product PI(3,4,5)P3 regulate eNOS subcellular localization in mammalian cells. In agreement with a role of PI3-kinase in regulating eNOS subcellular localization, several studies have shown that this kinase and its products are important for the regulation of protein trafficking in various cell types (7, 49). Specifically, PI3-kinase has been shown to be necessary in basolateral-to-apical trafficking of some proteins in cultured epithelial cells (24). Thus it is likely that PI3-kinase regulates eNOS activity not only by stimulating its phosphorylation but also by modulating eNOS subcellular localization in epithelial cells.
Activation of PI3-kinase has been reportedly involved in signal transduction initiated by mechanical stimulation in various cell types. However, the precise mechanism by which flow, shear stress, or stretch activates PI3-kinase is not fully understood. In general, PI3-kinase is activated by translocation to the plasma membrane after phosphorylation of transmembrane tyrosine kinase receptors and subsequent interaction of PI3-kinase SH2 domains with phosphotyrosine residues in the receptors (51). It has recently been shown that flow activates PI3-kinase by inducing Src kinase activation and subsequent ligand-independent activation of tyrosine kinase receptors such as the VEGF and purinergic P2 receptors (26, 45). Given that c-Src is expressed in the THAL (28), it is tempting to speculate that flow-dependent activation of c-Src mediates PI3-kinase activation and eNOS trafficking in the THAL.
Luminal flow has also been shown to induce a rapid increase in intracellular calcium in renal epithelial cells (41, 54). In endothelial cells, eNOS activation by flow is independent of calcium and calcium/calmodulin stimulation (11, 12, 50), arguing against a role for intracellular calcium in eNOS trafficking and activation in the THAL. However, in some cells mechanical stimulation increases intracellular calcium and activates PI3-kinase (8). Thus calcium may be involved in the regulation of eNOS trafficking and activation in the THAL indirectly by allowing maximal stimulation of PI3-kinase by flow. We are currently investigating the mechanism of PI3-kinase activation by flow in the THAL.
Very little is known about the role of PI3-kinase-stimulated eNOS activity along the nephron and its role in regulating salt and water reabsorption. Previous data from our laboratory showed that eNOS-derived NO inhibits NaCl absorption by the THAL (35, 38) and that NO produced in this nephron segment acts in a paracrine manner to modulate tubuloglomerular feedback (TGF) (52). We have also reported that hormones that inhibit NaCl absorption by the THAL may do so by activating the PI3-kinase/eNOS pathway (37). Thus regulation of eNOS by PI3-kinase in the THAL is likely to play an important physiological role as a regulator of NO levels in the renal medulla, thereby promoting salt and water excretion by the kidney. Impaired PI3-kinase signaling has been reported in pathological states such as diabetes (1, 3) and obesity (2, 56), and these conditions are associated with enhanced sodium retention and hypertension. Thus our data may identify a common defect in PI3-kinase signaling in these conditions that is linked to increased salt reabsorption by the loop of Henle.
Interaction between eNOS and the chaperone Hsp90 is well documented (13, 17, 44). In vitro experiments have shown that Hsp90 directly binds eNOS and that this interaction increases Akt-dependent phosphorylation of eNOS at serine 1179 (13). The activation of PI3-kinase by flow leads to Akt activation in endothelial cells (15), and in the companion study we observed increased phosphorylation of eNOS at serine 1179 in THALs (34a). Thus we studied whether Hsp90 is involved in flow-induced eNOS translocation and found that geldanamycin, a Hsp90 inhibitor, completely blocked flow-induced eNOS translocation in THALs. We also found that flow altered Hsp90 localization, inducing its translocation to the apical membrane; and, as with eNOS, Hsp90 translocation was completely blocked by PI3-kinase inhibition. While it is currently not possible to state the exact role of Hsp90 in eNOS translocation, it has been shown that Hsp90 regulates eNOS activity by two mechanisms. First, Hsp90 may act as a scaffolding protein, increasing the interaction of eNOS with Akt (13), and, second, Hsp90 may directly modify eNOS enzymatic activity by changing its structure via direct binding (48). While the exact mechanism by which Hsp90 influences eNOS activity is still unclear, our data suggest that besides these two mechanisms, Hsp90 may regulate eNOS activity by modulating its subcellular localization as shown for other proteins that interact with eNOS (59). Because Hsp90 is also involved in regulated trafficking of other proteins, such as the glucocorticoid receptor (6, 42, 43), it is possible that regulated trafficking of eNOS is dependent on the same protein machinery involved in Hsp90-mediated trafficking of the glucocorticoid receptor; however, this remains to be determined.
We have previously found that NO produced by eNOS inhibits NaCl and NaHCO3 absorption by the THAL (27–29, 31, 34). Thus it is likely that luminal flow, via eNOS activation, also regulates NaCl transport in this nephron segment. In addition, we have previously observed that NO produced by the THAL blunts TGF (44). Thus it is possible that flow-induced eNOS activation constitutes a feed-forward mechanism in which flow-induced NO blunts TGF, thereby decreasing afferent arteriole diameter, increasing glomerular filtration rate, and heightening flow along the nephron. However, the existence of this mechanism remains to be tested.
In addition to the THAL, eNOS is expressed in other epithelial cells such as the pulmonary epithelium (58), where NO stimulates ciliary beat frequency (27) and regulates the amount and composition of airway surface liquid. In the brain, eNOS has been localized to the ciliated epithelium of the ependymal cells lining the ventricles (58). In the testis, eNOS is localized to the seminiferous tubules and Leydig cells (58, 60), where its function is still unclear, whereas eNOS expressed in cervical epithelial cells is known to increase paracellular permeability and mucus secretion (21–23). To our knowledge, there are no data regarding regulation of eNOS in any of these cells; nevertheless, given that they are subjected to changes in luminal flow or mechanical stimulation, it is likely that PI3-kinase-dependent eNOS translocation and activation play a role in the physiology of these cells.
We conclude that flow-induced eNOS translocation and activation in the THAL are mediated by PI3-kinase and production of PI(3,4,5)P3 and that Hsp90 is involved in flow-induced eNOS translocation. We believe our data show for the first time that PI3-kinase regulates eNOS activity and subcellular localization in epithelial cells.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-28982 and HL-70985 (to J. L. Garvin).
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