published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

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Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Previous studies in our lab have shown that a subset of Eph family receptor tyrosine kinases, including EphA2, is strongly and persistently upregulated in renal tubular cells in both in vitro and in vivo models of the renal IRI. Src kinases are necessary and sufficient for upregulation of EphA2. We have proposed that IRI-induced EphA2 upregulation may serve as a necessary step in renal tubular remodelling. / In this study, we have further defined the mechanism of Src kinase-induced EphA2 upregulation by identifying the -145/+137 EphA2 promoter region as the minimal region required for basal and Src kinase-induced activation of the promoter. Moreover, we have identified within this region, at position -45, a canonical cAMP response element (CRE) (Nowakowski et al.), which is essential for EphA2 promoter activation. However, we also found that the prototypical CRE-binding transcription factor, CREB, was not necessary for activation of the EphA2 promoter, suggesting that CREB-related or -unrelated transcription factors are responsible for EphA2 upregulation.

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.

La información respecto a la respuesta del corazón aislado de ratas hipertensas espontáneas (SHR) a la isquemia-reperfusión y a los mecanismos de protección descriptos para ratas normotensas [pre y postacondicionamiento isquémicos (PI y PCI, respectivamente) y farmacológicos], es escasa. Por lo tanto, la participación de la vía PI3K/Akt/GSK-3β y del poro de permeabilidad transitoria de la mitocondria (PPTM) en los mecanismos responsables de la muerte ó la sobrevida de los cardiomiocitos de SHR, no están debidamente aclarados. El objetivo general de este trabajo de tesis fue estudiar en corazones de SHR aislados y perfundidos con la técnica de Langendorff los efectos de la isquemia global (IG, 45 min)-reperfusión (R, 60 min), del PI y PCI y del tratamiento con ClLi e IMI (inhibidores de GSK-3β) sobre el tamaño del infarto, el daño oxidativo, la sensibilidad del PPTM al Ca2+, la liberación de citocromo c al citosol y la ultraestructura mitocondrial. El protocolo de IG-R produjo un tamaño del infarto de aproximadamente 50% del área de riesgo y daño oxidativo, evidenciado por un aumento de la peroxidación lipídica (TBARS), una disminución marcada del contenido de GSH y un aumento de la actividad de SODT y SODMn. El contenido de P-GSK-3β y P-Akt y la sensibilidad del PPTM al Ca2+ disminuyeron, mientras que la expresión de citocromo c en el citosol aumentó. La microscopia electrónica reveló que la mayor parte de las mitocondrias estaban dañadas, con edema y destrucción de sus crestas. Las intervenciones PI y PCI y el tratamiento con los inhibidores de GSK-3β protegieron a los corazones de SHR de los daños antes mencionados. Por lo tanto, en los corazones intervenidos y tratados se observó una disminución del tamaño del infarto y del daño oxidativo evidenciado por la disminución de la peroxidación lipídica (TBARS), preservación parcial del contenido de GSH y disminución de la actividad de SODT y SODMn. La expresión de P-GSK3β y de P-Akt y de la sensibilidad del PPTM al Ca2+ aumentó mientras que el contenido de citocromo c en el citosol disminuyó. Por microscopia electrónica fue posible encontrar en estos grupos la presencia de algunas mitocondrias con ultraestructura conservada. Los efectos beneficiosos del PI y PCI fueron anulados cuando la vía de señalización de PI3K/Akt fue inhibida con wortmanina. Las variables mencionadas retornaron a los valores observados en los corazones isquémicos no tratados. De las relaciones examinadas surge que: a- el tamaño del infarto es mayor cuando la peroxidación lipídica aumenta y el contenido de GSH disminuye. En estas condiciones la sensibilidad del PPTM al Ca2+ es menor. La situación opuesta se da en presencia de intervenciones cardiorpotectoras. Por lo tanto, en ellas el tamaño del infarto es menor cuando la peroxidación lipídica disminuye y el contenido de GSH aumenta. En estas condiciones la sensibilidad del PPTM al Ca2+ tiende a recuperarse. En base a los datos obtenidos se concluye que las alteraciones de la formación y/ó apertura del PPTM, participan y determinan la muerte ó la sobrevida celular en el corazón hipertrófico de SHR sometido a isquemia-reperfusión. Así, la disminución del tamaño del infarto obtenida con las intervenciones y/ó tratamientos utilizados fue el resultado de la disminución del daño oxidativo íntimamente asociada a la recuperación parcial de la integridad mitocondrial -menor apertura del PPTM- vía P-Akt/P-GSK-3β. Otro hallazgo interesante fue que la protección por el tratamiento con ClLi (fármaco ampliamente utilizado en psiquiatría) fue similar a la obtenida con las intervenciones de acondicionamiento isquémico (PI y PCI). Por lo tanto, esta droga podría ser una posible herramienta terapéutica para atenuar la injuria por isquemia y reperfusión. / Information regarding to the response to ischemia-reperfusion in isolated hearts from spontaneously hypertensive rats (SHR) as well as the effect of the protective mechanisms described in normotensive rats (ischemic and pharmacological pre and postconditioning, IP and IPC), is scarce. Therefore, the participation of PI3K/Akt/GSK-3β pathway and the mitochondrial permeability transition pore (mPTP) in the mechanisms responsible for cardiomyocyte death or survival are not properly clarified. The objective of this investigation was to study in isolated hearts from SHR the effects of global ischemia (GI, 45 min) and reperfusion (R, 60 min), and the actions of IP, IPC, treatments with LiCl and IMI (GSK-3β inhibitors) on infarct size, oxidative damage, mPTP Ca2+ sensitivity, cytochrome c release to the cytosol and mitochondrial ultrastructure. The protocol of GI-R produced an infarct size of approximately 50%, increased oxidative damage as evidenced by an increase of lipid peroxidation (TBARS), a decrease of GSH content and an increase of Total SOD and MnSOD activity. The content of P-GSK-3β and P-Akt and the mPTP Ca2+ sensitivity decreased while the expression of cytochrome c in the cytosol increased. Electron microscopy showed that most of the mitochondria were damaged, presenting edema and destruction of the cristae. IP, IPC and pharmacologic treatments with both GSK-3β inhibitors protected the hearts against reperfusion injury. Therefore, in the hearts treated we observed a smaller infarct size and reduced oxidative damage (decreased lipid peroxidation (TBARS), partial preservation of GSH and decreased Total SOD and MnSOD activity) compared to non-treated ischemic hearts. The P-GSK-3β and P-Akt expression and mPTP Ca2+ sensitivity increased while the cytosolic cytochrome c content decreased. By electron microscopy it was possible to find some mitochondria with normal ultrastructure. The beneficial effects of IP and IPC were canceled when the PI3K/Akt was inhibited with wortmannin. All the parameters examined returned to the values observed in non-treated ischemic hearts. Analyzing the relationships: Infarct size vs. TBARS and GSH, and mPTP Ca2+ sensitivity vs. Infarct size and TBARS, it arises that: the Infarct size increased when lipid peroxidation increased and GSH content decreased. Under these conditions the mPTP Ca2+ sensitivity decreased. The opposite situation occurs in the presence of the cardioprotective interventions. Therefore, the infarct size decreased when lipid peroxidation decreased and GSH content increased. Under these conditions the mPTP Ca2+ sensitivity tended to recover. Based on the data obtained, we suggest that the alterations of formation and/or opening of the mPTP participate and determine cell death or survival in the hypertrophic heart of SHR subjected to ischemia-reperfusion. Thus, the reduction in infarct size obtained with the ischemic interventions and /or treatments derived from the decreased oxidative damage intimately linked to a partial recovery of mitochondrial integrity- less mPTP opening- via P-Akt/P-GSK-3β. Another interesting finding was that the protection by the treatment with LiCl (drug widely used in psychiatry) was similar to that obtained with ischemic interventions (IP and IPC). Therefore, this drug emerges as a potential therapeutic tool in reducing the post-ischemic changes.

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.