Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

Abstract

Anaerobic mitochondrial metabolism of ␣-ketoglutarate and aspartate or ␣-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules. 34 The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130 cas migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to ϳ5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of ␤1 and ␣6 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. ␣-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 ␣-ketoglutarate and aspartate or ␣-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. Ischemic and related forms of acute renal failure result from a complex interplay of vascular and tubular events that can vary in their relative contributions depending on characteristics of the specific clinical situation or experimental model. Although the process is frequently also termed acute tubular necrosis, much of the tubule cell damage in both animal models and human acute renal failure is sublethal and reversible within affected cells. [1][2][3][4][5][6] Effects on multiple subcellular structures have been described including loss of brush border microvilli and simplification of the basolateral membrane, 1-3 disruption of the normal polar distribution of major membrane-associated proteins including Na ϩ ,K ϩ -ATPase and its associated cytoskeletal proteins, 7,8 disruption of tight junctions and adherens junctions, 5,9 -13 and abnormalities of integrin distribution and function, 14 -19 all of which can contribute to impaired barrier function and vectorial transport by the epithelium. ATP depletion and the resulting protein dephosphorylation 10 -13,20,21 are the primary processes initiating these events.Proximal tubules have relatively little or no glycolytic capacity making them dependent on mitochondrial metabolism for ATP synthesis. 22,23 Freshly isolated proximal tubules rapidly develop lethal damage when subjected to hypoxia or other ATP-depleting maneuvers, 24 -27 which has limited their utility for studying reversible structural and metabolic alterations. This situation has improved with recognition that much of their se...

Tolerance of the Human Kidney to Isolated Controlled Ischemia

“…These findings dramatically contrast to those seen in animal models, which showed greater severity and extent of these changes after being subjected to shorter durations of ischemia than were most patients in our cohort. 31,32,35,36 Consistent with the limited apical structural damage, basal b1 integrin, which is highly sensitive to ischemia in animal models and to hypoxia of isolated tubules, 21,22 was almost unaffected. In maximally hypoxic isolated tubules, immunodetectable phosphotyrosine is entirely lost and then recovers during reoxygenation as a function of ATP availability.…”

“…Tubule injury produced by ischemia or hypoxia is sensitively reflected by alterations of actin and integrins. 21,22 Cellular ATP depletion decreases protein tyrosine phosphorylation. 22,23 Intercellular adhesion molecule-1 (ICAM-1) is an important early mediator of the inflammatory component of ischemic AKI.…”

“…21,22 Cellular ATP depletion decreases protein tyrosine phosphorylation. 22,23 Intercellular adhesion molecule-1 (ICAM-1) is an important early mediator of the inflammatory component of ischemic AKI. 5,24 Therefore, we used these indices to further assess injury produced by ischemia, as well as to confirm The frequency with which they changed was similar to that seen for actin ( Figure 5).…”

Heat Shock Protein 27 Is the Major Differentially Phosphorylated Protein Involved in Renal Epithelial Cellular Stress Response and Controls Focal Adhesion Organization and Apoptosis

“…Signaling mediated by the focal adhesion protein complex is essential in various cellular processes, including cell migration, proliferation, and survival. In vitro studies in freshly isolated tubules as well as primary cultured renal PTC and renal cell lines indicate an essential role of focal adhesionmediated signaling in the molecular mechanism of PTC injury, which involves disruption of the focal adhesions and a reorganization of the F-actin network (2). Cellular stress results in activation of MAPK family members.…”

Focal Adhesion Kinase Signaling Mediates Acute Renal Injury Induced by Ischemia/Reperfusion

“…I/R injury induces alterations in the F-actin cytoskeleton 8 and redistribution of integrins 3 and other FA-associated signaling proteins. 5 Disruption of adhesion was associated with dephosphorylation of FA proteins during hypoxia in isolated tubules 9 and I/R injury in vivo. 5 The effect of disruption of FA-mediated signaling events on the pathogenesis of acute renal failure is however still unclear.…”