Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules
2001; Elsevier BV; Volume: 158; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64687-1
ISSN1525-2191
AutoresJoel M. Weinberg, Manjeri A. Venkatachalam, Nancy F. Roeser, Ruth A. Senter, Itzhak Nissim,
Tópico(s)Cellular Mechanics and Interactions
ResumoAnaerobic mitochondrial metabolism of α-ketoglutarate and aspartate or α-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130cas migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to ∼5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of β1 and α6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. α-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of α-ketoglutarate and aspartate or α-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia. Anaerobic mitochondrial metabolism of α-ketoglutarate and aspartate or α-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130cas migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to ∼5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of β1 and α6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. α-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of α-ketoglutarate and aspartate or α-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia. 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ATP depletion and the resulting protein dephosphorylation10Mandel LJ Bacallao R Zampighi G Uncoupling of the molecular ‘fence' and paracellular ‘gate' functions in epithelial tight junctions.Nature. 1993; 361: 552-555Crossref PubMed Scopus (247) Google Scholar, 11Tsukamoto T Nigam SK Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins.Am J Physiol. 1999; 276: F737-F750PubMed Google Scholar, 12Tsukamoto T Nigam SK Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly.J Biol Chem. 1997; 272: 16133-16139Crossref PubMed Scopus (177) Google Scholar, 13Schwartz JH Shih T Menza SA Lieberthal W ATP depletion increases tyrosine phosphorylation of beta-catenin and plakoglobin in renal tubular cells.J Am Soc Nephrol. 1999; 10: 2297-2305Crossref PubMed Google Scholar, 20Kobryn CE Mandel LJ Decreased protein phosphorylation induced by anoxia in proximal renal tubules.Am J Physiol. 1994; 267: C1073-C1079PubMed Google Scholar, 21Chen J Cohn JA Mandel LJ Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.Proc Natl Acad Sci USA. 1995; 92: 7495-7499Crossref PubMed Scopus (104) Google Scholar are the primary processes initiating these events. 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This situation has improved with recognition that much of their sensitivity is because of the formation of pathological plasma membrane pores that can be blocked by glycine at physiological levels.28Weinberg JM Davis JA Abarzua M Rajan T Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules.J Clin Invest. 1987; 80: 1446-1454Crossref PubMed Scopus (262) Google Scholar, 29Dong Z Patel Y Saikumar P Weinberg JM Venkatachalam MA Development of porous defects in plasma membranes of ATP-depleted Madin-Darby canine kidney cells and its inhibition by glycine.Lab Invest. 1998; 78: 657-668PubMed Google Scholar By suppressing this type of plasma membrane damage, replacement of glycine allows examination of specific injury mechanisms in vitro, uncomplicated by the plethora of postmortem degenerative changes that would otherwise occur in multiple cellular systems.30Weinberg JM Venkatachalam MA Goldberg H Roeser NF Davis JA Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells.Am J Physiol. 1995; 268: F110-F121PubMed Google Scholar, 31Nurko S Sogabe K Davis JA Roeser NF Defrain M Chien A Hinshaw D Athey B Meixner W Venkatachalam MA Weinberg JM Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury.Am J Physiol. 1996; 270: F39-F52PubMed Google Scholar, 32Sogabe K Roeser NF Davis JA Nurko S Venkatachalam MA Weinberg JM Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.Am J Physiol. 1996; 271: F292-F303PubMed Google Scholar We have found that, despite glycine cytoprotection, freshly isolated, kidney proximal tubule cells develop a profound mitochondrial functional deficit during hypoxia/reoxygenation that is characterized by incomplete recovery of energization during reoxygenation, impaired respiration for substrates dependent on function of electron transport complex I, partial de-energization, and persistence of mitochondrial matrix condensation.33Weinberg JM Roeser NF Davis JA Venkatachalam MA Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.Kidney Int. 1997; 52: 140-151Crossref PubMed Scopus (43) Google Scholar, 34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar The mitochondrial lesion can be substantially ameliorated and recovery of cell ATP strikingly enhanced by supplementing the tubules with α-ketoglutarate plus aspartate (αKG/ASP), α-ketoglutarate plus malate (αKG/MAL), or other specific citric acid cycle metabolites during either hypoxia or reoxygenation.34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar The substrates promote mitochondrial pathways of anaerobic metabolism to increase ATP production by substrate level phosphorylation and energization by anaerobic respiration in electron transport complexes I and II34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar and provide succinate to bypass the complex I block when aerobic metabolism resumes.35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar Two related observations in these initial studies34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar spurred us to further investigate the relationships between anaerobic ATP generated in mitochondria and its utilization by cells to effect repair and survival. First, we were struck by the relatively small yields of energy by anaerobic metabolism of mitochondrial substrates in proximal tubules as assessed by absolute levels of ATP attained during hypoxia, ie, increases from 4.3% of oxygenated control levels in tubules without protective substrates at the end of 60 minutes hypoxia to 6.9% with the substrates.34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar Second, we noted that the ability to perform complex-integrated functions was regained to a remarkable degree during reoxygenation of substrate supplemented hypoxic tubules as indicated by increases of ATP concentrations and respiratory rates. Similar degrees of recovery could be documented in tubules that had been provided with anaerobic mitochondrial substrates only during reoxygenation.34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar These observations suggest that the low levels of additional ATP generation during hypoxia or early reoxygenation made possible by the substrates were sufficient for utilization to maintain or repair structure by cells that were otherwise committed to continuing ATP depletion and a lethal outcome.35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar In the present studies we have assessed the impact of the tubule energetic deficit and its modification by protective substrates on the alterations of cytoskeletal and focal adhesion protein distribution and tyrosine phosphorylation during hypoxia/reoxygenation. Cellular interactions with the extracellular matrix are essential to maintain the integrity of signal transduction pathways that ensure survival.36O'Brien V Frisch SM Juliano RL Expression of the integrin a5 subunit in HT29 colon carcinoma cells suppresses apoptosis triggered by serum deprivation.Exp Cell Res. 1996; 224: 208-213Crossref PubMed Scopus (85) Google Scholar, 37Ilic D Almeida EA Schlaepfer DD Dazin P Aizawa S Damsky CH Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.J Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (440) Google Scholar, 38Chan PC Lai JF Cheng CH Tang MJ Chiu CC Chen HC Suppression of ultraviolet irradiation-induced apoptosis by overexpression of focal adhesion kinase in Madin-Darby canine kidney cells.J Biol Chem. 1999; 274: 26901-26906Crossref PubMed Scopus (101) Google Scholar Moreover, the phosphorylation states of these proteins could serve as indices of the availability of mitochondrially generated, anaerobic ATP at peripheral sites. The data provide evidence that ATP generated by the protective substrates during hypoxia is available in the cytosol and that the mitochondrial recovery promoted by the substrates during reoxygenation plays a pivotal role to enable a strikingly complete rephosphorylation of tyrosine-phosphorylated proteins and coordinated re-assembly of focal adhesions that is not otherwise achieved. They indicate a major effect of early energetic recovery on critical cell-matrix interactions and the potential for enhancing these interactions with specific citric acid cycle metabolites that optimize mitochondrial function. Proximal tubules were prepared from kidney cortex of female New Zealand White rabbits (1.5 to 2.0 kg; Oakwood Farms, Oakwood, MI) by digestion with combinations of Worthington Type I (Worthington, Freehold, NJ) and Sigma blend type H or F collagenase and centrifugation on self-forming Percoll gradients as described.28Weinberg JM Davis JA Abarzua M Rajan T Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules.J Clin Invest. 1987; 80: 1446-1454Crossref PubMed Scopus (262) Google Scholar, 30Weinberg JM Venkatachalam MA Goldberg H Roeser NF Davis JA Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells.Am J Physiol. 1995; 268: F110-F121PubMed Google Scholar, 31Nurko S Sogabe K Davis JA Roeser NF Defrain M Chien A Hinshaw D Athey B Meixner W Venkatachalam MA Weinberg JM Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury.Am J Physiol. 1996; 270: F39-F52PubMed Google Scholar, 32Sogabe K Roeser NF Davis JA Nurko S Venkatachalam MA Weinberg JM Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.Am J Physiol. 1996; 271: F292-F303PubMed Google Scholar Incubation conditions generally followed our published protocols.28Weinberg JM Davis JA Abarzua M Rajan T Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules.J Clin Invest. 1987; 80: 1446-1454Crossref PubMed Scopus (262) Google Scholar, 32Sogabe K Roeser NF Davis JA Nurko S Venkatachalam MA Weinberg JM Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.Am J Physiol. 1996; 271: F292-F303PubMed Google Scholar, 33Weinberg JM Roeser NF Davis JA Venkatachalam MA Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.Kidney Int. 1997; 52: 140-151Crossref PubMed Scopus (43) Google Scholar Tubules were suspended at 3.0 to 5.0 mg of tubule protein/ml in a 95% O2/5% CO2-gassed solution (medium A) containing (in mmol/L): 110 NaCl, 2.6 KCl, 25 NaHCO3, 2.4 KH2PO4, 1.25 CaCl2, 1.2 MgCl2, 1.2 MgSO4, 5 glucose, 4 sodium lactate, 0.3 alanine, 5.0 sodium butyrate, 3% dialyzed dextran (T-40, Pharmacia), and 2 glycine. Medium A was also supplemented with 0.5 mg/ml of bovine gelatin (75 bloom) to suppress aggregation of the isolated tubules during the prolonged experimental incubation periods. After 15 minutes of preincubation at 37°C, tubules were resuspended in fresh medium A with experimental agents as needed and regassed with either 95% O2/5% CO2 (controls) or 95% N2/5% CO2 (hypoxia). The bicarbonate concentration of medium A during hypoxia was decreased to maintain a pH of 6.9 to simulate tissue acidosis during ischemia in vivo.33Weinberg JM Roeser NF Davis JA Venkatachalam MA Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.Kidney Int. 1997; 52: 140-151Crossref PubMed Scopus (43) Google Scholar After 60 minutes, samples were removed for analysis. The remaining tubules were washed twice to remove any experimental substrates being tested for their efficacy only during hypoxia and then resuspended in fresh 95% O2/5% CO2-gassed, pH 7.4 medium A with additional experimental substrates as needed. For the reoxygenation period, sodium butyrate in medium A was replaced with 2.0 mmol/L sodium heptanoate and, to maximize availability of purine precursors for ATP resynthesis, 250 μmol/L AMP or ATP was added in most experiments.33Weinberg JM Roeser NF Davis JA Venkatachalam MA Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function.Kidney Int. 1997; 52: 140-151Crossref PubMed Scopus (43) Google Scholar After 60 minutes of reoxygenation, samples were removed again for analysis. Cell ATP was measured and ultrastructural studies done as previously described.32Sogabe K Roeser NF Davis JA Nurko S Venkatachalam MA Weinberg JM Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli.Am J Physiol. 1996; 271: F292-F303PubMed Google Scholar Sampling and analysis for other parameters was as in the following sections. For staining with the carbocyanine dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazocarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR),34Weinberg JM Venkatachalam MA Roeser NF Nissim I Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.Proc Natl Acad Sci USA. 2000; 97: 2826-2831Crossref PubMed Scopus (259) Google Scholar, 35Weinberg JM Venkatachalam MA Roeser NF Saikumar P Dong Z Senter RA Nissim I Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.Am J Physiol. 2000; 279: F927-F943Google Scholar, 39Chen LB Smiley ST Probing mitochondrial membrane potential in living cells by a J-aggregate-forming dye.in: Mason WT Fluorescent and Luminescent Probes for Biological Activity. Academic Press, New York1994: 124-132Google Scholar an aliquot from a 1000× stock solution in dimethyl sulfoxide was mixed with an equal volume of serum, dispersed as an intermediate 100× stock solution in phosphate-buffered saline (PBS), and then added to a final concentration of 5 μg/ml in the tubule suspension at the end of the desired experimental period. The suspension was regassed with O2/CO2 and incubated in the dark for an additional 15 minutes at 37°C, then tubules were washed three times in an ice-cold solution containing (in mmol/L): 110 NaCl, 25 NaHEPES, pH 7.2, 1.25 CaCl2, 1.0 MgCl2, 1.0 KH2PO4, 3.5 KCl, 5.0 glycine, and 5% polyethylene glycol (average molecular weight 8000). A 300-μl aliquot of the washed tubules containing 1.2 to 1.5 mg of protein was brought up to 2.5 ml with additional ice cold wash solution and then scanned during continuous gentle stirring using a Photon Technology International (Monmouth Junction, NJ) Alphascan fluorometer at 488 nm excitation/500 to 620 nm emission. Under these conditions, the peak of the green fluorescence of the monomeric form of the dye was at 530 nm and the red fluorescence of the J-aggregates peaked at 590 nm. Tubules were pelleted, then dispersed in ice-cold, 10.0 mmol/L sodium imidazole, pH 7.15, 10 mmol/L sodium-EGTA, 1.0% Triton X-100, 5.0 μg/ml leupeptin, 5.0 μg/ml pepstatin A, 5.0 μg/ml aprotinin, 1.0 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 50 mmol/L sodium fluoride, 20 mmol/L β-glycerol phosphate, 1 mmol/L sodium orthovanadate, 12 μmol/L cyclosporine A (Calbiochem, San Diego, CA), and 12 nmol/L calyculin (Calbiochem). The protein was then immediately precipitated in four volumes of ice-cold methanol and dissolved in 2% sodium dodecyl sulfate/10% glycerol/0.125 mol/L Tris-HCl, pH 7.4, then stored at −80°C until analysis. To optimize detection of β1 integrin by immunoblotting, samples for it were collected by resuspending pelleted tubules in boiling 1% sodium dodecyl sulfate, 1 mmol/L sodium orthovanadate, 10 mmol/L Tris-HCl, pH 7.4, and boiled for 5 minutes. Protein concentrations were measured using bicinchoninic acid (Pierce, Rockford, IL) with bovine serum albumin as the standard. For immunoblotting, samples were mixed 1:1 with a double-strength running buffer containing 0.12 mol/L Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.01% bromophenol blue, and 2% β-mercaptoethanol, boiled for 5 minutes, run on precasted 4 to 12% gradient polyacrylamide gels (Novex, San Diego, CA), and then transferred onto nitrocellulose filters. For studies of β1 integrin, β-mercaptoethanol was omitted from the running buffer. Most proteins were immunodetected using mouse monoclonal antibodies (mAbs) as the primaries (usually 1 μg/ml) and peroxidase-conjugated goat anti-mouse (Pierce) as the secondary followed by chemiluminescence (ECL; Amersham, Arlington Heights, IL). Anti-pY mAbs were from either Upstate Biotechnology, Lake Placid, NY, (4G10), or Cell Signaling Technology, Beverly, MA, (P-Tyr-102, agarose-conjugated P-Tyr-100). mAbs to paxillin (clone 349), the 85-kd α-chain of PI-3-kinase (clone 4), focal adhesion kinase (clone 77), and p130cas(clone 21) were from Transduction Laboratories (Lexington, KY). mAb to the β1 integrin subunit was from Chemicon (LM534; Temecula, CA). Phosphorylation state-specific polyclonal rabbit antibodies to pY31Nurko S Sogabe K Davis JA Roeser NF Defrain M Chien A Hinshaw D Athey B Meixner W Venkatachalam MA Weinberg JM Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury.Am J
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