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Am J Physiol Renal Physiol 289: F1103-F1115, 2005. First published May 24, 2005; doi:10.1152/ajprenal.00458.2004
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Acute renal ischemia rapidly activates the energy sensor AMPK but does not increase phosphorylation of eNOS-Ser1177

Peter F. Mount,1,2,3 Rebecca E. Hill,1 Scott A. Fraser,1,3 Vicki Levidiotis,1,2,3 Frosa Katsis,4 Bruce E. Kemp,4,5 and David A. Power1,2,3

1Austin Research Institute, 2Department of Nephrology and 3Department of Medicine, Austin Health, University of Melbourne, Heidelberg, Victoria; 4St. Vincent’s Institute, Fitzroy, Victoria; and 5Commonwealth Scientific and Industrial Research Organisation Health Sciences and Nutrition, Parkville, Victoria, Australia

Submitted 20 December 2004 ; accepted in final form 19 May 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A fundamental aspect of acute renal ischemia is energy depletion, manifest as a falling level of ATP that is associated with a simultaneous rise in AMP. The energy sensor AMP-activated protein kinase (AMPK) is activated by a rising AMP-to-ATP ratio, but its role in acute renal ischemia is unknown. AMPK is activated in the ischemic heart and is reported to phosphorylate both endothelial nitric oxide synthase (eNOS) and acetyl-CoA carboxylase. To study activation of AMPK in acute renal ischemia, the renal pedicle of anesthetized Sprague-Dawley rats was cross-clamped for increasing time intervals. AMPK was strongly activated within 1 min and remained so after 30 min. However, despite the robust activation of AMPK, acute renal ischemia did not increase phosphorylation of the AMPK phosphorylation sites eNOS-Ser1177 or acetyl-CoA carboxylase-Ser79. Activation of AMPK in bovine aortic endothelial cells by the ATP-depleting agent antimycin A and the antidiabetic drug phenformin also did not increase phosphorylation of eNOS-Ser1177, confirming that AMPK activation and phosphorylation of eNOS are dissociated in some situations. Immunoprecipitation studies demonstrated that the dissociation between AMPK activation and phosphorylation of eNOS-Ser1177 was not due to changes in the physical associations between AMPK, eNOS, or heat shock protein 90. In conclusion, acute renal ischemia rapidly activates the energy sensor AMPK, which is known to maintain ATP reserves during energy stress. The substrates it phosphorylates, however, are different from those in other organs such as the heart.

AMP-activated protein kinase; endothelial nitric oxide synthase; acetyl-CoA carboxylase; kidney

ACUTE RENAL FAILURE IS A COMMON condition occurring in 5% of all hospitalized patients and in up to 25% of those who are critically ill (41). Despite advances in renal replacement therapy, mortality of patients with acute renal failure remains high at ~50% (41). The most common cause of acute renal failure is acute tubular necrosis, which frequently occurs in situations of reduced renal perfusion and renal ischemia (29). Common causes include hypotension, such as occurs with sepsis or hypovolemia, renal transplantation, and thrombotic or embolic disease of the renal arteries. The basic physiological consequence of acute renal ischemia is energy depletion, manifest as falling levels of ATP, which is associated with a concomitant rise in the level of AMP (21, 47, 48, 52). The energy sensor AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that has a central role in coordinating cellular energy metabolism (24). AMPK is activated in situations of energy stress and phosphorylates multiple substrates to both inhibit ATP-consuming pathways, such as protein and lipid synthesis, and to stimulate pathways leading to ATP production, such as fatty acid {beta}-oxidation and glycolysis (8, 24, 30). Thus activation of AMPK acts to maintain ATP reserves in the face of energy stress. In the ischemic heart, activation of AMPK has been demonstrated to have a protective role, as mice with hearts that express a kinase dead form of AMPK have more severe ATP depletion, increased apoptotic cell death, and impaired recovery of left ventricular function following ischemia-reperfusion (43).

We (15) recently described expression of AMPK in the normal rat kidney, where the {alpha}1-catalytic subunit was found to be highly expressed, whereas the {alpha}2-isoform was barely detectable. In rats, renal AMPK activity was regulated by dietary salt (15); however, the role of AMPK in acute renal ischemia has not been studied. Furthermore, nothing is known about the substrates that are phosphorylated by AMPK in the kidney and whether these are similar to or different from those that have been described in other organs such as heart, liver, and skeletal muscle. AMPK is activated in the ischemic heart, where it is reported to phosphorylate endothelial nitric oxide synthase (eNOS) (11), acetyl-CoA carboxylase (ACC) (27), 6-phosphofructo-2-kinase (30), and eucaryotic elongation factor 2 kinase (22). In the ischemic heart, AMPK is reported to stimulate NO production by phosphorylating eNOS at Ser1177 (11). Regulation of eNOS-Ser1177 phosphorylation is complex and involves at least four other kinases, as well as regulatory molecules such as heat shock protein 90 (Hsp90) (7). In addition to cardiac ischemia, AMPK has also been implicated in the regulation of eNOS-Ser1177 phosphorylation in response to other stimuli such as adiponectin and peroxynitrite (9, 55). AMPK also phosphorylates eNOS at Thr495 in vitro, which has the effect of reducing eNOS activity (11); however, there is presently no evidence that AMPK can phosphorylate eNOS-Thr495 in vivo. ACC phosphorylation at Ser79 occurs simultaneously with activation of AMPK in severe cardiac ischemia, although ACC phosphorylation is not increased during mild cardiac ischemia, despite activation of AMPK (2). In the heart, phosphorylation of ACC by AMPK stimulates fatty acid {beta}-oxidation (44). In contrast, in the liver, ACC phosphorylation by AMPK both reduces fatty acid synthesis and increases fatty acid oxidation (42).

Here, we report the first study of AMPK in the ischemic kidney and determine that AMPK is strongly activated within 1 min of the onset of acute renal ischemia. However, despite the activation of AMPK, there was no increase in phosphorylation of the AMPK substrates eNOS and ACC. The dissociation between AMPK activation and phosphorylation of eNOS-Ser1177 was also observed in an in vitro model of endothelial cell ischemia using the ATP-depleting agent antimycin A and in response to AMPK activation by the antidiabetic drug phenformin. The dissociation between AMPK activation and phosphorylation of eNOS-Ser1177 was not due to changes in the level of physical association between AMPK, eNOS, or Hsp90.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. Rabbit polyclonal antibodies against {alpha}1-AMPK, {alpha}2-AMPK, pThr172 {alpha}-AMPK, p-ACC-Ser79, p-eNOS-Ser1177, p-eNOS-Ser633, p-eNOS-Ser615, and p-eNOS-Thr495 were produced as previously described (11, 12, 32). All antibodies were affinity purified. Antibodies against phosphopeptides were purified using the corresponding phosphopeptide affinity columns after preclearing with dephosphopeptide affinity columns. The specificity of the purified antibodies was evaluated by both enzyme immunoassay and immunoblotting before use in experiments.

Mouse monoclonal antibodies against eNOS and Hsp90 were purchased from BD Transduction Laboratories (Lexington, KY).

Secondary antibodies [swine anti-rabbit horseradish peroxidase (HRP), rabbit anti-mouse HRP, streptavidin-HRP] were purchased from Dako (Carpinteria, CA). Protein A-HRP was purchased from Amersham Pharmacia (Uppsala, Sweden).

Acute renal ischemia model. Male Sprague-Dawley rats (250–300 g) (Western Australian Research Facility) were anesthetized by intraperitoneal injection of a combination of xylazine (7.5 mg/kg; Troy Laboratories) and ketamine (60 mg/kg; Purnell Laboratories) administered as a single dose. Once anesthesia was established, a midline laparotomy was performed and the left kidney was exposed and gently mobilized. Acute renal ischemia was induced by cross-clamping the renal pedicle for various time points (0 s, 10 s, 1 min, 5 min, 10 min, 30 min) before freeze clamping the kidney in situ. Freeze clamping in situ was performed with metal tongs that were precooled in liquid nitrogen. The frozen kidney was then immersed in liquid nitrogen and stored at –80°C until required for analysis. Tissue for immunofluorescence was immediately immersed for fixation in 4% paraformaldehyde. All studies were approved by and performed in accordance with the guidelines of The Animal Ethics Committee of Austin Health.

To prepare homogenates, each frozen kidney was placed in 5 ml of ice-cold homogenization buffer (50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 30 mM sodium pyrophosphate, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS) and homogenized immediately by glass homogenization with 50 strokes in a Dounce homogenizer. Protease inhibitors were added at the following concentrations: 2 mM PMSF, 1 mM benzamidine, 0.02 mg/ml trypsin inhibitor, 0.02 mg/ml leupeptin, and 0.02 mg/ml aprotinin (all protease inhibitors were purchased from Sigma-Aldrich, St. Louis, MO). Homogenates were rotated at 4°C for 1 h before centrifugation to ensure complete membrane solubilization and then centrifuged at 16,000 g for 5 min at 4°C; the resulting pellet containing cell nuclei and tissue debris was discarded. Supernatants were then recentrifuged at 100,000 g for 60 min at 4°C in an ultracentrifuge for removal of cytoskeletal proteins. The protein concentration of the supernatant was determined using the Bradford method (Bio-Rad protein assay kit). Homogenates were stored at –80°C until required.

Cell culture. Bovine aortic endothelial cells (BAECs) were passaged from primary cultures and used for experiments between passages 3 and 10. The cells were cultured in flasks, and dishes were coated with 1% gelatin in DMEM (5.6 mM glucose) supplemented with 10% FCS, 20 µg/ml cis-hydroxyproline, 60 µg/ml penicillin, and 500 µg/ml streptomycin. Experiments were performed on cells grown to confluence in 9-cm-diameter plastic dishes. Because various factors in serum are reported to regulate both AMPK and eNOS activity, stimulations were performed under serum-free conditions. Two hours before the addition of antimycin A, phenformin, bradykinin, or sorbitol (all purchased from Sigma-Aldrich), the culture medium was changed to DMEM (5.6 mM glucose) without FCS. The concentration and duration of exposure for the various stimuli are stated with the actual results. Cell lysis was performed with 0.5 ml of ice-cold cell lysis buffer (50 mM HEPES, pH 7.5, 2 mM EDTA, 50 mM NaF, 5 mM Na4P2O7, 1 mM DTT, 1% Nonidet P-40, 10 µg/ml trypsin inhibitor, 10 µg/ml aprotinin, 1 mM PMSF).

Immunoprecipitations and partial purification of eNOS by ADP-Sepharose. AMPK was immunoprecipitated from kidney homogenate and BAEC lysate by mixing 4 µl of {alpha}1-AMPK antibody (1 mg/ml) with homogenate or lysate at 4°C for 1 h. eNOS was immunoprecipitated from BAEC lysate by adding 5 µl of anti-eNOS monoclonal antibody (BD Transduction Laboratories) (250 µg/ml) at 4°C for 1 h. Immunocomplexes were collected by mixing the sample with protein A- or protein G-Sepharose beads (20 µl; Amersham Pharmacia) at 4°C for 30 min. ADP-Sepharose pulldowns to partially purify eNOS from kidney homogenates and BAEC lysates were performed by mixing 20 µl of 2',5'-ADP-Sepharose beads (Amersham Pharmacia) with the homogenate or lysate sample at 4°C for 90 min as previously described (10). Immunoprecipitations and ADP-Sepharose pulldowns from kidney homogenates were performed with 10 mg of total protein. Immunoprecipitations and ADP-Sepharose pulldowns from BAEC lysates were performed with 0.5–1 mg of total protein. After immunoprecipitation or ADP-Sepharose pulldown, the beads were collected by centrifugation and washed three times in ice-cold wash buffer (1% Nonidet P-40 in PBS) and once in cold PBS. Reducing Laemmli sample buffer (20 µl) was added to the beads, which were then heated to 95°C for 4 min before SDS-PAGE separation and Western blot analyses.

Western blotting. Samples were separated by SDS-PAGE and electrically transferred to a polyvinidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) at 30 V overnight. The membrane was blocked in 5% casein in Tris-buffered saline (TBS) for 1 h and then incubated in primary antibody. The optimal antibody concentration and duration of incubation were determined for each antibody. After washing in TBS-0.05% Tween 20, the membrane was incubated for 30 min in secondary antibody (swine anti-rabbit-HRP or rabbit anti-mouse-HRP, Dako; or protein A-HRP, Amersham) at 1/2,500 dilution. After a further washing in TBS-0.05% Tween 20, immunoreactive proteins were detected by enhanced chemiluminescence with the SuperSignal chemiluminescent system (Pierce). If the membrane was to be probed with another primary antibody, antibody bound to the membrane was stripped by incubation in 1x reblot stripping solution (Chemicon, Temecula, CA) for 15 min. Quantification of Western blots was by densitometry (Scion Image for Windows; Scion, Frederick, MD).

AMPK activity assay. AMPK activity in kidney homogenate was measured as previously described (15). Briefly, AMPK was immunoprecipitated from 10 mg of kidney homogenates, and then a phosphorylation reaction was performed in kinase assay buffer to measure AMPK activity: 50 mM HEPES, pH 7.5, 10 mM MgCl2, 5% glycerol, 1 mM DTT, 0.05% Triton X-100, 250 µM [{gamma}-32P]ATP (500 cpm/pmol), and 100 µM ADR-1 peptide substrate (33).

Immunofluorescence. Immunofluorescence staining for activated AMPK was performed using the rabbit polyclonal antibody specific for the AMPK {alpha}-subunit phosphorylated at Thr172. Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, cut in sections 4 µm thick, and dewaxed. Antigen retrieval was performed by microwave treating the sections in citrate buffer (pH 6) for 15 min. After being washed in PBS, the sections were permeabilized [0.3% Triton X-100 and 0.025% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in PBS] for 30 min. Sections were incubated in 150 mM glycine for 20 min to quench aldehyde groups and then blocked in 3% BSA with 0.03% Triton X-100 and 0.025% CHAPS for 20 min. Primary antibody was diluted at 1:100 in blocking buffer and incubated on sections overnight at 4°C. Control sections were incubated in rabbit polyclonal antibody against hemaglutinin at 1:100 (Santa Cruz, La Jolla, CA). Sections were then washed in PBS, and anti-rabbit Alexa 488 (Molecular Probes, Eugene, OR) diluted 1:150 in blocking buffer was applied to sections for 30 min. Sections were then washed three times in PBS (3 min each) and coverslipped using Dako fluorescent mounting medium. Sections were examined by fluorescence microscopy, and images were captures using Leica DC Viewer software (version 3.2.0.0 [EC] , 2000). The signal-to-noise ratio was adjusted in the nonischemic control sections to give minimal background fluorescence and then kept constant at the same level while all other images were captured.

Statistics. We used Instat version 3.05 (GraphPad Software, San Diego, CA) for statistical analyses. Data are presented as means ± SE. Unless otherwise stated, data were analyzed by ANOVA; if significant, Bonferroni’s tests for multiple comparisons were used. P values <0.05 were deemed significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of AMPK, eNOS, and ACC to the soluble fraction of kidney homogenates. As described in MATERIALS AND METHODS, kidney homogenates were prepared in homogenization buffer containing 1% Nonidet P-40, 0.25% sodium deoxycholate, and 0.1% SDS and then centrifuged at 16,000 g for 5 min to remove cell nuclei and debris and again at 100,000 g for 60 min to remove remaining insoluble elements. In the above detergents, it was predicted that AMPK, eNOS, and ACC would be found in the supernatant. To determine the actual distributions of AMPK, eNOS, and ACC, Western blots were performed on both supernatant and pellet fractions (Fig. 1). Under both control and ischemic (5 min) conditions, all three molecules were present in the supernatant but not in the insoluble pellet produced by the 100,000-g spin (Fig. 1). There was also no detectable AMPK, eNOS, or ACC in the pellet produced by the first spin (data not shown). Therefore, all other analyses of AMPK, eNOS, and ACC in kidney homogenates were performed on the soluble fraction.



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  		Fig. 1. Analyses of supernatant and pellet fractions for presence of AMP-activated protein kinase (AMPK), endothelial nitric oxide synthase (eNOS), and acetyl-CoA carboxylase (ACC). Control and ischemic (5 min) kidneys were homogenized and processed as described in MATERIALS AND METHODS in homogenization buffer containing 1% Nonidet P-40, 0.1% SDS, and 0.25% deoxycholate. After centrifugation at 100,000 g for 60 min, proteins from both the supernatant (S) and pellet (P) fractions were suspended in Laemelli sample buffer containing 2% SDS and separated by SDS-PAGE. Protein concentration was measured by the Bradford assay, and 50 µg of protein were loaded in all lanes. Antibodies against {alpha}1-AMPK (A) and eNOS (B) were used to determine expression by Western blot as described in MATERIALS AND METHODS. Detection of ACC (C) was determined by Western blot using streptavidin-horseradish peroxidase (HRP), which recognizes biotinylated proteins, including ACC1 (255 kDa) and pyruvate carboxylase (PC) (130 kDa). Polyvinidene difluoride membranes were stained with Ponceau S to confirm equivalent protein loading in each lane.

 
Activation of AMPK by acute renal ischemia. The effect of acute renal ischemia on the activity of AMPK was analyzed by both Western blot and AMPK activity assay in kidneys subjected to acute ischemia for increasing time intervals (0 s, 10 s, 1 min, 5 min, 10 min, 30 min) (Fig. 2). Immunoprecipitations of AMPK were performed using an antibody specific for the {alpha}1-subunit, as we have previously determined that the normal rat kidney (Sprague-Dawley) contains the {alpha}1 catalytic subunit but not the {alpha}2 (15). No increase in AMPK activity was evident after 10 s of ischemia by either method. After 1 min of ischemia, however, Thr172-{alpha} phosphorylation was increased 8.6-fold (P < 0.001), and AMPK activity (ADR-1 phosphorylation) was increased 1.8-fold (P < 0.001 by two-tailed unpaired t-test). AMPK activity was further increased after 5 min of ischemia, whether measured by Thr172-{alpha} phosphorylation (18.2-fold increase, P < 0.001) or by activity assay (2.7-fold increase, P < 0.01). Increasing the duration of ischemia to 10 or 30 min did not produce any greater activation of AMPK compared with that seen at 5 min; however, AMPK was still markedly activated at these time points compared with baseline (0 s) (Fig. 2).



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  		Fig. 2. Activation of AMPK in the kidney by acute renal ischemia. For A and B, ischemia was induced for increasing time intervals and AMPK was immunoprecipitated from 10 mg of kidney homogenate using an antibody against the {alpha}1-subunit and analyzed for activated (pThr172-{alpha}) and total ({alpha}1) AMPK by Western blot. A: representative Western blots. B: cumulative densitometry data (arbitrary units) showing the effects of acute renal ischemia on Thr172-{alpha} phosphorylation. C: AMPK was immunoprecipitated from 10 mg of kidney homogenate using an antibody against the {alpha}1-subunit and AMPK activity was quantified by measuring phosphorylation of the AMPK peptide substrate ADR-1. Time on x-axes is measured in min (') and s (''). For B and C, n = 8 at 0 s. For all other time points, n = 4. Results are means ± SE. **P < 0.01, *P < 0.001 compared with 0 s by ANOVA using the Bonferroni multiple comparison test.

 
Localization of increased renal AMPK activity during renal ischemia. To determine whether the activation of AMPK by acute renal ischemia was diffuse or restricted to specific regions or structures of the kidney, immunofluorescence microscopy was performed using the polyclonal antibody against the activated form (pThr172-{alpha}) of AMPK. In nonischemic kidneys, there was increased fluorescence, consistent with basally activated AMPK, at the apical membranes of specific tubules (Fig. 3A). We have previously shown that this basal pThr172-{alpha}-AMPK staining in the normal rat kidney is predominantly at the apical membranes of cortical thick ascending limbs and macula densa (15). The basolateral staining of collecting ducts that was variably observed in our previous study was not seen in this study. After 5 min of ischemia, there was a marked increase in the intensity and extent of the apical staining (Fig. 3C). In addition, there was a diffuse increase in pThr172-{alpha} fluorescence throughout all of the cortical tubules, as well as in glomeruli (Fig. 3C). The increased fluorescence with ischemia was specific for the pThr172-{alpha} antibody, as it was not seen with a rabbit polyclonal antibody against an irrelevant epitope (hemaglutinin) (Fig. 3E). Interestingly, however, there was no increase in pThr172-{alpha} fluorescence observed in the inner medulla (Fig. 3D). To ensure pThr172-{alpha}-AMPK antibody specificity and an absence of cross reactivity with other phosphorylated proteins, Western blots were performed on whole kidney homogenate from control and ischemic kidneys. Ischemia caused a marked increase in pThr172-{alpha}-AMPK with no other major bands being seen (data not shown).



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  		Fig. 3. Localization of activated AMPK during acute renal ischemia by pThr172-{alpha} immunofluorescence. Control (A and B) and 5-min ischemic (C–F) kidneys were paraformaldehyde fixed, and immunofluorescence was performed for activated AMPK (pThr172-{alpha}). Anti-hemaglutinin (HA) rabbit polyclonal antibody was used as a negative control. Images were captured using Leica DC viewer software. The signal-to-noise ratio was kept constant during the capture of images. Bar (in F) = 50 µm.

 
Effect of renal ischemia on phosphorylation of eNOS. Because AMPK is reported to stimulate NO synthesis by phosphorylation of eNOS-Ser1177, the effect of AMPK activation by acute renal ischemia on phosphorylation at this site was studied. In addition, the effect of renal ischemia on phosphorylation of eNOS-Thr495 was also studied. AMPK has been described as phosphorylating eNOS-Thr495 in vitro (11); however, there are so far no reports of AMPK phosphorylating this site in vivo. In contrast to the effect of phosphorylation of Ser1177, phosphorylation of eNOS-Thr495 reduces eNOS activity. In this study, the principal finding was that, despite the activation of AMPK, acute renal ischemia did not regulate phosphorylation of eNOS at either Ser1177 or Thr495 (Fig. 4A).



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  		Fig. 4. Effect of acute renal ischemia on phosphorylation of eNOS and ACC. A: eNOS was partially purified by ADP-Sepharose pulled down from 10 mg of kidney homogenate and phosphorylations of eNOS-Ser1177 and eNOS-Thr495 were determined by Western blot. B: samples of homogenate (75 µg) from kidneys ischemic for various time intervals were analyzed for ACC phosphorylation by Western blot. ACC phosphorylation at Ser79 was detected by a rabbit polyclonal antibody. Total ACC expression was detected using streptavidin-HRP. Both eNOS and ACC phosphorylations were quantified by densitometry. For baseline phosphorylation (0 s), n = 8. For all other time points, n = 4. Results are means ± SE.

 
Effect of renal ischemia on phosphorylation of ACC. ACC exists as two isoforms designated as ACC1 and ACC2, both of which are recognized substrates of AMPK. Phosphorylation of ACC1 at Ser79 by AMPK turns off fatty acid synthesis (18). ACC2 is localized to the mitochondria and is essential for the control of fatty acid oxidation, which is stimulated by AMPK phosphorylation of ACC2 (10). By Western blot, only ACC1 was detectable in the kidney (Fig. 4B). Basal phosphorylation of ACC1-Ser79 was detectable in the nonischemic (0 min) control kidneys (Fig. 4B). Despite the activation of AMPK, renal ischemia did not further increase phosphorylation of ACC1-Ser79 (Fig. 4B).

Effect of in vitro endothelial cell ischemia on AMPK activity and eNOS phosphorylation. Several studies have now reported that AMPK can stimulate NO synthesis by phosphorylation of eNOS-Ser1177 (9, 11, 37, 55). Therefore, the finding that eNOS-Ser1177 phosphorylation was not increased in the ischemic kidney, despite a large increase in AMPK activity, was unexpected. To further study the relationship between AMPK activation and eNOS phosphorylation during ischemia, a model of in vitro endothelial cell ischemia was studied using BAECs. BAECs were chosen because they endogenously express AMPK and eNOS. In the kidney, both endothelial and tubular cells are reported to express eNOS, although the highest level of expression is in endothelium (3).

Before study of AMPK activation in BAECs, expression of the catalytic ({alpha}) subunits of AMPK expressed in BAECs was determined by immunoprecipitation and Western blot (Fig. 5A). BAECs were found to express the {alpha}1-subunit but not the {alpha}2-subunit. Therefore, in subsequent experiments, immunoprecipitations of AMPK and Western blots for AMPK expression from BAECs were performed using the antibody against the {alpha}1-subunit. As stated in the methods, BAECs were placed in serum-free DMEM for 2 h before stimulation, to exclude possible influence of factors in serum on phosphorylation of AMPK, eNOS, and ACC. In unstimulated BAECS, low levels of basal phosphorylation of AMPK, eNOS, and ACC could be detected and was not altered by the presence or absence of FCS (Fig. 5B).



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  		Fig. 5. Analysis of AMPK catalytic ({alpha}) subunit expression in bovine aortic endothelial cells (BAECs) and the effect of experimental conditions on basal phosphorylation of AMPK, eNOS, and ACC. A: to determine the isoforms of the catalytic ({alpha}) subunits expressed in BAECs, immunoprecipitations (IP) were separately performed for both the {alpha}1- and {alpha}2-subunits from 1 mg of BAEC lysate protein. As a positive control, immunoprecipitations were also performed from 1 mg of rat heart homogenate. Negative control immunoprecipitations were performed with polyclonal rabbit immunoglobulin. Resulting immunoprecipitates were then analyzed by Western blot for expression of the {alpha}1- and {alpha}2-subunits. B: confluent BAECS were incubated in serum-free DMEM or fresh DMEM containing 10% FCS for 2 h and then lysed. AMPK was immunoprecipitated by an antibody against the {alpha}1-subunit, and eNOS was pulled down by ADP-Sepharose. Western blots of ACC were on total cell lysate. Phosphorylations of AMPK, eNOS, and ACC were determined by Western blot as before.

 
Using a previously established method (49), we mimicked ischemia in BAECs by ATP depletion using the respiratory chain inhibitor antimycin A (0.1 µM). Antimycin A caused a marked activation of AMPK, as evidenced by a greater than sevenfold increase in Thr172-{alpha} phosphorylation (P < 0.01), that was maximal after 5–10 min (Fig. 6A). However, exposure of BAECs to 0.1 µM antimycin A did not increase phosphorylation at the AMPK phosphorylation site eNOS-Ser1177 (Fig. 6B). Phosphorylation of eNOS-Thr495 was also not altered in response to ATP depletion by antimycin A (Fig. 6C). More recently, regulation of eNOS has been described by phosphorylation at Ser615 and Ser633; however, there is no recognized role for AMPK in regulation of phosphorylation of these sites (32). Phosphorylations of eNOS at Ser615 and Ser633 were also not regulated in response to antimycin A (Fig. 6C).



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  		Fig. 6. Effect of ATP depletion by antimycin A on AMPK activity and eNOS phosphorylation in BAECs. A: BAECS were subjected to ATP depletion by incubation with 0.1 µM antimycin A for increasing time intervals. AMPK was immunoprecipitated using an antibody against the {alpha}1-subunit, and activity was measured by Western blot for the activated form phosphorylated at Thr172{alpha}. Results were quantified by densitometry (n = 4, *P < 0.01). B: after ATP depletion for increasing time intervals, eNOS-Ser1177 phosphorylation was analyzed after ADP-Sepharose pulldown and Western blot. Results were quantified by densitometry (n = 6). C: effect of 0.1 µM antimycin A on phosphorylation of eNOS at Ser1177, Ser633, Ser615, and Thr495. Blots shown in C represent 3 similarly performed experiments.

 
Effect of activation of AMPK by phenformin and sorbitol on phosphorylation of eNOS-Ser1177. Although AMPK is activated by stimuli such as ischemia and antimycin A, which increase the AMP-to-ATP ratio, other stimuli such as metformin and sorbitol also activate AMPK without a measurable increase in the AMP-to-ATP ratio (16). However, these apparently AMP-independent activators do act by stimulation of AMPK phosphorylation in the catalytic loop at Thr172-{alpha} (16). To determine whether the activation of AMPK by AMP-independent stimuli was associated with stimulation of phosphorylation of eNOS-Ser1177, BAECs were incubated with the metformin analog phenformin (2 mM, 30 min) and sorbitol (600 mM, 30 min). Bradykinin (1 µM, 5 min), a well-described agonist for eNOS-Ser1177 phosphorylation (19), was used as a positive control for eNOS phosphorylation.

Consistent with their known effects, phenformin, sorbitol, and antimycin A all significantly activated AMPK, as shown by a sixfold or greater increase in Thr172-{alpha} phosphorylation (P < 0.05) (Fig. 7A). Interestingly, bradykinin also caused a significant activation of AMPK (P < 0.05) (Fig. 7A). In each case, activation of AMPK caused a corresponding increase in phosphorylation of ACC-Ser79 of at least 13-fold (P < 0.05) (Fig. 7C). No effect on eNOS-Ser1177 phosphorylation was seen following activation of AMPK by either phenformin or, as seen earlier, antimycin A (Fig. 7B). Sorbitol, however, caused a marked increase (9.1-fold) in phosphorylation of eNOS-Ser1177 (P < 0.05) (Fig. 7B).



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  		Fig. 7. Effect of activating AMPK in BAECs by phenformin and sorbitol on phosphorylation of eNOS and ACC. Confluent BAECs were stimulated with bradykinin (1 µM, 5 min), antimycin A (0.1 µM, 10 min), phenformin (2 mM, 30 min), and sorbitol (600 mM, 30 min). AMPK was immunoprecipitated with an antibody against the {alpha}1-subunit, and eNOS was pulled down by ADP-Sepharose. Phosphorylation and expression of AMPK (A) and eNOS (B) were then analyzed by Western blot. Phosphorylation of ACC (C) was analyzed by Western blot of total cell lysate. Results were quantified by densitometry and expressed as arbitrary units, showing fold increase compared with unstimulated BAECs (n = 4, *P < 0.05 compared with control). D: phosphorylation of AMPK and eNOS were also analyzed by Western blot of 25 µg of total cell lysate to determine whether the immunoprecipitated AMPK and pulled-down eNOS were representative of AMPK and eNOS in the whole cell. Blots shown in D represent 3 similarly performed experiments.

 
Because AMPK and eNOS were analyzed after immunoprecipitation and ADP-Sepharose pulldown, respectively, it might have been possible that this analysis did not represent all of the cellular AMPK and eNOS. To test this possibility, phosphorylations of AMPK and eNOS were reanalyzed by Western blot of whole cell lysates (Fig. 7D). With this method, the effect of each of the activators on AMPK and eNOS phosphorylation was the same as already demonstrated.

Effect of ATP depletion in BAECs on associations between eNOS, AMPK, and Hsp90. Several studies have reported that eNOS coimmunoprecipitates with the catalytic ({alpha}) subunit of AMPK (11, 28, 55). To determine whether the lack of increase in eNOS-Ser1177 phosphorylation after stimulation by antimycin A was explained by a change in the level of physical association between AMPK and eNOS, eNOS was immunoprecipitated after cell lysis, and Western blots were performed for both phosphorylation and expression of eNOS and AMPK (Fig. 8A). Bradykinin (1 µM, 5 min) was used as a positive control for eNOS phosphorylation. In vitro ischemia of BAECs caused by antimycin A (0.1 µM, 10 min) did not alter the level of coimmunoprecipitation between eNOS and AMPK, as shown by immunoprecipitation with antibodies against both eNOS (Fig. 8A) and AMPK (Fig. 8B). Moreover, the level of activation of AMPK, as determined by Thr172-{alpha} phosphorylation, was as strongly increased in the AMPK that associated with eNOS (Fig. 8A) as it was in the rest of the cell (Fig. 8B).



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  		Fig. 8. Effect of ATP depletion by antimycin A on the associations between eNOS, AMPK, and heat shock protein 90 (Hsp90). BAECs were stimulated with vehicle (control), antimycin A (0.1 µM, 10 min), and bradykinin (1 µM, 5 min). A: eNOS was immunoprecipitated using a monoclonal antibody against eNOS (BD Transduction Laboratories). Immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blot for phosphorylation and expression of eNOS. Total ({alpha}1) and activated (Thr172-{alpha}) AMPK that coimmunoprecipitated with eNOS were also detected by Western blot. Results shown represent 3 similarly performed experiments. B: AMPK was immunoprecipitated using a rabbit polyclonal antibody against the {alpha}1-subunit. Immunoprecipitates were subjected to SDS-PAGE and analyzed for phosphorylation and expression of AMPK. Coimmunoprecipitation of total eNOS was determined by Western blot. Results shown represent 3 similarly performed experiments. C: eNOS was immunoprecipitated using a monoclonal antibody against eNOS (BD Transduction Laboratories). Immunoprecipitates were subject to SDS-PAGE and analyzed by Western blot for phosphorylation and expression of eNOS and coimmunoprecipitated Hsp90. Results shown represent 3 similarly performed experiments.

 
Hsp90 is an ATP-dependent chaperone protein that is reported to have a role in activation and phosphorylation of eNOS (17). Because Hsp90 is ATP dependent, we investigated whether antimycin A altered the degree of coimmunoprecipitation between eNOS and Hsp90, which might then affect the ability of kinases to phosphorylate eNOS-Ser1177. Antimycin A (0.1 µM, 10 min), however, did not alter the amount of Hsp90 that coimmunoprecipitated with eNOS (Fig. 8C).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This study demonstrates, for the first time, that acute renal ischemia initiates a rapid and sustained activation of the energy sensor AMPK within 1 min of ischemia onset, with peak activity occurring within 5–10 min. Activation of AMPK is mediated primarily by binding of AMP to the Bateman domains of the {gamma}-subunit (1, 45). As well as having a direct allosteric effect, AMP binding further increases AMPK activity by enhancing phosphorylation of AMPK at its T-loop residue (Thr172-{alpha}) by one or more upstream AMPK-kinases (20). Previous studies in kidneys have demonstrated that ATP levels are reduced by 70% after 1 min and 90% after 7 min of acute ischemia, and this is associated with a simultaneous rise in AMP (21, 47). Interestingly, however, low levels of ATP at 5–10% of baseline are maintained during acute renal ischemia for 30–60 min (13). Recently, the tumor suppressor LKB1 was identified as an important upstream AMPK-kinase that phosphorylates Thr172-{alpha} in the AMPK catalytic loop (54). There may, however, be other AMPK-kinases; for example, a recent study found that myocardial ischemia increased AMPK-kinase activity that was not due to LKB1 activation, but the alternate AMPK-kinase was not identified (2). Thus the activation of AMPK observed in the present study is consistent with an effect mediated by an increased AMP-to-ATP ratio. Whether the upstream AMPK-kinase mediating phosphorylation of Thr172-{alpha} is LKB1 or another AMPK-kinase is unknown.

Using immunofluorescence microscopy, we observed a diffuse increase in AMPK activity in the cortex after 5 min of ischemia. However, no increase was seen in the inner medulla. The tubules found in the inner medulla are the descending and ascending thin limbs of Henle’s loop and inner medullary collecting ducts. These tubules have a significantly lower Na+-K+-ATPase activity and, therefore, energy requirement, than tubules found in the outer medulla and cortex, including proximal convoluted tubules, thick ascending limbs, and distal convoluted tubules (23). Therefore, our finding might be explained by the fact that the AMP-to-ATP ratio would be slower to increase with ischemia in the tubules found in the inner medulla. We acknowledge that absolute verification of the specificity of these immunofluorescence changes requires demonstration of the absence of staining in double AMPK {alpha}1 and {alpha}2 knockout tissue; however, such tissue is not available. Nonetheless, the pattern of increasing AMPK activity with acute renal ischemia that was seen by immunofluorescence parallels that also demonstrated by AMPK activity assay and by Western blots of both immunoprecipitated AMPK and whole kidney homogenates.

We have previously demonstrated that AMPK directly phosphorylates eNOS in vitro at the activating site Ser1177 and the inhibitory site Thr495 (11). Furthermore, activation of AMPK in the heart by ischemia was associated with an increase in eNOS-Ser1177 phosphorylation, whereas Thr495 phosphorylation was unchanged (11). Since this original observation, activation of AMPK has also been associated with increased phosphorylation of eNOS-Ser1177 in response to peroxynitrite, adiponectin, the AMP mimetic 5-aminoimidazole 4-carboxamide riboside (AICAR) and during angiogenesis in response to hypoxia (9, 34, 35, 55). We predicted, therefore, that activation of AMPK in the ischemic kidney would stimulate NO synthesis by phosphorylation of eNOS-Ser1177. Such an effect would be predicted to be beneficial in terms of energy balance, as NO both stimulates vasodilatation and inhibits the energy-expensive process of tubular sodium reabsorption (36). The present study, however, shows that activation of AMPK in the ischemic kidney is not associated with any change in phosphorylation of eNOS at Ser1177 or Thr495.

Because the dissociation between AMPK activation and eNOS-Ser1177 phosphorylation was unexpected, further studies were performed using an in vitro model of ischemia in BAECs. With the use of ATP-depleting agent antimycin A, there was a strong and rapid activation of AMPK without any increase in phosphorylation of eNOS-Ser1177 or changes in other eNOS phosphorylation sites. Immunoprecipitation studies demonstrated that the dissociation between AMPK activation and eNOS-Ser1177 phosphorylation was not due to changes in physical association between AMPK and eNOS. Indeed, strongly activated AMPK was coimmunoprecipitated with eNOS from BAECs that had been ATP depleted with antimycin A.

Dissociation between AMPK activation and eNOS-Ser1177 phosphorylation was also observed following activation of AMPK by the antidiabetic metformin analog phenformin. The mechanism of activation of AMPK by phenformin is unclear, but it is likely to be similar to that of metformin, which activates AMPK without altering the AMP-to-ATP ratio (16). In contrast, bradykinin and sorbitol both activated AMPK and increased eNOS-Ser1177 phosphorylation; however, these two phenomena are not necessarily causally related. In fact, stimulation of eNOS-Ser1177 phosphorylation in response to bradykinin has previously been attributed in different studies to the kinases Akt, calmodulin-dependant kinase II, and protein kinase A (4, 14, 19). Clearly, further studies are required to determine which of the kinases that can phosphorylate eNOS-Ser1177 is most important in mediating the effect of bradykinin. Activation of AMPK by bradykinin has previously been reported in Chinese hamster ovary cells stably transfected with the bradykinin B2 receptor (26). Notably, however, the present study is the first to describe stimulation of AMPK by bradykinin in nontransfected cells and to show that this occurs in endothelium. Regarding the effect of sorbitol on phosphorylation of eNOS-Ser1177, hyperosmotic stress is known to activate numerous kinases, including Akt and mitogen-activated protein kinases (5, 51). Simultaneous activation of AMPK and the increase in eNOS-Ser1177 phosphorylation following sorbitol stimulation, therefore, might not be directly linked.

Myocardial ischemia (11), the AMP mimetic AICAR (34), peroxynitrite (55), and adiponectin (9) have all been reported to increase phosphorylation of eNOS-Ser1177 phosphorylation in an AMPK-dependent manner. The observation that acute renal ischemia did not increase phosphorylation of eNOS-Ser1177, despite strong activation of AMPK, was therefore unexpected. Our subsequent experiments in BAECs stimulated by antimycin A and phenformin also found no increase in eNOS-Ser1177 phosphorylation, despite clear activation of AMPK. Thus an important implication of the present study is that the relationship between AMPK activation and eNOS-Ser1177 phosphorylation varies with different stimuli and in different tissues. Curiously, in the case of antimycin A, the dissociation between AMPK activation and phosphorylation of eNOS-Ser1177 occurred even though activated AMPK could be coimmunoprecipitated with eNOS. The concept that AMPK activation and eNOS-Ser1177 phosphorylation is dissociated in some situations is supported by a recent study by Shearer et al. (46), which found that infusion of AICAR did not increase phosphorylation of eNOS-Ser1177 in skeletal muscle of rats that were also given L-NAME or intralipid, despite definite activation of AMPK.

Analogous to our findings for the relationship between AMPK and eNOS-Ser1177 phosphorylation, regulation of eNOS-Ser1177 phosphorylation by the kinase Akt is also known to vary with different stimuli (50). Takahashi et al. (50) observed that, although insulin and PDGF both strongly activated Akt in BAECs, insulin caused a marked increase in eNOS-Ser1177 phosphorylation but PDGF had no effect. This observation was explained by the finding that insulin recruited the chaperone protein Hsp90 and this facilitated Akt phosphorylation, whereas Hsp90 recruitment did not occur with PDGF (50). Because Hsp90 is an ATP-dependant molecule (38), we determined whether ATP depletion with antimycin A could disrupt the association between eNOS and Hsp90. No effect was seen. It is possible that the action of another molecule might be required when AMPK phosphorylates eNOS-Ser1177; however, the identity of such a proposed molecule is presently unknown.

Activation of AMPK by acute renal ischemia also did not increase phosphorylation of ACC-Ser79. This was also unexpected because several studies have reported that AMPK phosphorylates ACC to inhibit fatty acid synthesis and increase fatty acid oxidation (42). Furthermore, our own observations confirm that activation of AMPK in BAECs by antimycin A, phenformin, sorbitol, and bradykinin all increase phosphorylation of ACC-Ser79. Significantly, however, Altarejos et al. (2) recently reported that mild cardiac ischemia had no effect on ACC phosphorylation despite an approximate doubling of AMPK activity. In the same study, a relatively minor increase in ACC phosphorylation was seen during severe cardiac ischemia, when AMPK activity was more than doubled (2). In addition, Wojaszweski et al. (53) reported dissociation between activation of AMPK and phosphorylation of ACC in human skeletal muscle during prolonged exercise. In the normal kidney, we observed significant basal phosphorylation of ACC1-Ser79. This is consistent with the low basal rate of fatty acid synthesis that occurs in the normal kidney (6). Given the absence of ACC2, the high basal rate of fatty acid oxidation of the normal kidney (39) does not appear regulated by AMPK. Importantly, when the effect of acute renal ischemia on fatty acid oxidation was studied, fatty acid oxidation was shown to be actually reduced (40), the opposite of what would be predicted if activation of AMPK regulated fatty acid oxidation in the ischemic kidney.

In summary, this study demonstrates that the energy-sensor AMPK is rapidly and strongly activated during acute renal ischemia. In the ischemic heart, the net effect of AMPK activation appears beneficial (43). In contrast, a recent study suggested that activation of AMPK in the ischemic brain worsens injury (31). Further studies are required to determine the functional effect of AMPK activation with kidney ischemia. The substrates phosphorylated by AMPK during renal ischemia remain to be determined, but clearly they differ from those seen in the ischemic heart. An intriguing consideration is that the major energy-consuming process in the kidney is tubular reabsorption of sodium by active transport (25); interestingly, we (15) have previously found that renal AMPK activity is increased in rats fed a high-salt diet. Given this, the possibility that AMPK phosphorylates kidney specific sodium transporters during ischemia is an attractive hypothesis for future study.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Health and Medical Research Council of Australia (NHMRC) Grants to D. A. Power and B. E. Kemp. S. A. Fraser is a Dora Lush Postgraduate Scholar of the NHMRC, P. F. Mount is a Medical Postgraduate Scholar of the NHMRC, V. Levidiotis is a Clinical Research Fellow of the NHMRC, and B. E. Kemp is an Australian Research Council Federation Fellow.


   ACKNOWLEDGMENTS
 
This work has been reported in abstract form at the American Society of Nephrology, Annual Scientific Meeting, St Louis, MO, October 2004 (J Am Soc Nephrol 15: SA-PO738, 2004).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. F. Mount, Austin Research Institute, Kronheimer Bldg., Austin Hospital, Studley Road, Heidelberg 3084, Victoria, Australia (e-mail: p.mount{at}ari.unimelb.edu.au)

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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