Browsing by Subject "Biochemical Phenomena, Metabolism, and Nutrition"
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Item Intravenous Pyruvate to Prevent Renal Injury Following Cardiac Arrest and Resuscitation(2014-08-01) Hollrah, Roger A.; Robert T. Mallet; Myoung-Gwi Ryou; Rong MaIntroduction: Cardiac arrest followed by resuscitation and recovery of spontaneous circulation (ROSC) produces systemic ischemia reperfusion (I/R), affecting all internal organs, including the kidney. This type of stress generates both a robust increase in reactive oxygen and nitrogen species (RONS) and an intense inflammatory response, which can result in renal cell death. The glycoprotein erythropoietin (EPO) has been shown to combat renal I/R injury by offering cyto-protection against inflammation and oxidative damage, as well as inhibiting apoptosis. The endogenous intermediary metabolite pyruvate has been observed to stabilize specific genetic machinery responsible for the production of EPO. This study was conducted to test the efficacy of intravenous pyruvate in exploiting these endogenous mechanisms of EPO to protect the kidney from cardiac arrest-induced, I/R injury. Hypothesis: Pyruvate administration during cardiopulmonary resuscitation (CPR), defibrillation, and ROSC will protect the kidneys from I/R injury by suppressing oxidative stress and inflammation via increased EPO production at the renal corticomedullary border. Methods: Yorkshire swine underwent 10 minutes of cardiac arrest, CPR effected by precordial compressions, and defibrillation, and were recovered for either 4 hours (acute) or 3 days (chronic). The animals were randomly assigned to 1 of 4 groups. Two groups underwent the cardiac arrest protocol described above: one group received intravenous infusion of 2M sodium pyruvate at a rate of 0.1 mmol∙kg-1∙min-1 during CPR and the first 60 minutes of recovery; the other group received an equimolar infusion of NaCl. The other two groups were surgically prepared and infused with NaCl or sodium pyruvate, but were not subjected to cardiac arrest, CPR, or defibrillation. For the acute protocol (n=28), animals were sacrificed 4hr after cardiac arrest, while in the chronic protocol (n=18), animals recovered for 3d before sacrifice. To evaluate the impact of cardiac arrest and pyruvate treatment on renal metabolism and antioxidant defense, proteins were extracted from snap-frozen renal corticomedullary border tissue for spectrophotometric activity assays of a panel of 10 metabolic and antioxidant enzymes; myeloperoxidase (MPO), an enzyme marker of pro-inflammatory leukocytes, was analyzed to assess inflammation. Plasma was sampled before cardiac arrest and at the time of biopsy to measure creatinine concentration, an indirect measure of glomerular filtration rate (GFR). Enzyme-linked immunosorbent assay (ELISA) kits were used to measure EPO content and Kidney Injury Molecule-1 (KIM-1) content, a receptor expressed on renal tubular cells that plays an important role in apoptosis. Tissue sections were stained with hematoxylin and eosin (H&E) and examined under light microscopy to count neutrophils and monocytes and to compare structure integrity across the different treatment groups and protocols. Results: In this study global I/R stress imposed on the kidneys by reversible cardiac arrest did not appreciably alter the activity of the 10 panel enzymes. Despite having no histological evidence of neutrophil infiltration (H&E stained slides), an increase in renal MPO activity was evident at 4 h recovery in the NaCl group which was prevented by pyruvate treatment (P [less than] 0.05). There was no evidence of ultrastructural damage to renal cortical and outer medullary structures. There was a noticeable increase in renal EPO content at 4 h ROSC vs. the sham group. An apparent, albeit not statistically significant, increase in KIM-1 content was observed in the two CPR groups vs. the NaCl-infused sham group. Plasma creatinine concentrations did not change appreciably between pre-arrest baseline and 3 d recovery. Interpretation and Conclusion: The I/R stress produced by the present cardiac arrest-resuscitation failed to alter appreciably the activities of the 10 panel enzymes, suggesting the oxidative stress was not sufficient to overwhelm the kidney’s endogenous antioxidant defenses. Plasma creatinine concentrations were also stable, implying the GFR was maintained and the glomerular ultrastructures were unaffected by I/R. The increase in MPO activity at 4 h ROSC implied a transient infiltration of inflammatory leukocytes, although none were visible on histological examination. The increase in KIM-1 content, though not statistically significant, suggests modest renal apoptotic activity after cardiac arrest and reperfusion. The transient increase in renal EPO content in the NaCl-infused post-arrest vs. sham pigs supports the possibility that even a brief period of renal ischemia by cardiac arrest can evoke renal EPO production. Collectively, these results indicate the renal I/R imposed by cardiac arrest and resuscitation does not inflict appreciable damage on the kidneys or its enzyme systems, at least within the first 3 d of post-arrest recovery. Abbreviations: AKI: acute kidney injury; ARF: acute renal failure; CK: creatine kinase; CPR: cardiopulmonary resuscitation; CS: citrate synthase; EPO: erythropoietin; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; G6PDH: glucose 6-phosphate dehydrogenase; GFR: glomerular filtration rate; GP: glutathione peroxidase; GR: glutathione reductase; HIF-1: hypoxia-inducible factor 1; I/R: ischemia-reperfusion; KIM-1: kidney injury molecule 1; LDH: lactate dehydrogenase; MPO: myeloperoxidase; PFK: phosphofructokinase; PHD: prolyl hydroxylase; RONS: reactive oxygen and nitrogen species; ROSC: recovery of spontaneous circulation.Item Study of Cross Bridge Kinetics in Hypertrophic Ventricular Muscle(2009-05-01) Muthu, Priya; Borejdo, JulianCardiovascular diseases are the leading cause of mortality worldwide; with heart failure being highly prevalent in most affluent parts of the world. There is a need for a better understanding of the mechanism underlying these diseases. Familial hypertrophic cardiomyopathy (FHC), one such disease, is a genetic disorder of the heart characterized by increased growth or hypertrophy in the thickness of the wall of the left ventricle, the largest of the four chambers of the heart. This research project is focused on one kind of FHC, the D166V mutation in the regulatory light chain in myosin, which is associated with a particularly malignant form of the disease. The overall goal of this project was to study cross bridge kinetics (contraction and ATP utilization) in cardiac muscle from transgenic mice and to develop assays to apply this to human samples. The real time orientation changes of myosin and actin during a single cross bridge cycle beginning in a state of rigor was studied by Fluorescence anisotropy. Rabbit psoas fibers were isolated and used to achieve imaging of a few fluorophores or cross bridges. This technique was then applied to study cardiac myofibrils from transgenic mice, carrying the mutation causing the disease (FHC). Methods to achieve single molecule detection to aid studying human samples suffering from this disease were developed using silver island films, monolayers of nanoparticles and surface plasmon coupled emission. The conclusions of this dissertation were that a mutation in a light chain in myosin cause changes in the cross bridge kinetics. Myofibrils from the mutated mice displayed a significant slower rate of detachment during contraction as well as increased ATPase activity, which if severe enough could cause the heart to compensate by increasing wall thickness (hypertrophy). Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. No therapy currently exists to treat or delay progression from hypertrophy to heart failure. This proposal help answer an important question regarding the molecular basis of FHC-mediated pathology in the heart. Also, achieving imaging of a single fluorophore has numerous implications in the biological field, like studying ligand-receptor interactions in live cells, involvement of protein molecules in internalization of bacteria by cells, monitoring the conformational fluctuations of DNA, diagnosis of prion diseases and also in detection of viruses at an early phase of infection.Item Synergy 2010: Annual Research Report(2010-01-01)Item Synergy 2011: Annual Research Report(2011-01-01)Item The Role of Glycogen Synthase Kinase-3β in the Regulation of Mitochondrial Membrane Permeability(2014-12-01) Brooks, Morgan M.; Patrick R. CammarataLens epithelial cells in a fully mature lens thrive in a hypoxic environment by developing several pro-survival mechanisms that can prevent cellular dysfunction. Many of these mechanisms focus on maintaining mitochondrial membrane integrity. Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient in a process termed mitochondrial membrane permeability transition (mMPT). The project herein focuses primarily on understanding the role of glycogen synthase kinase-3β (GSK-3β) in preventing mMPT in human lens epithelial (HLE-B3) cells; and, understanding that role in relation to extracellular signal-regulated kinase 1/2 (ERK1/2), a known regulator of GSK-3β activity. These studies further define mitoprotective mechanisms of lens cells by identifying how ERK1/2 and GSK-3β can directly (through the mitochondrial transition pore) or indirectly (through the induction of apoptosis) effect mitochondrial membrane potential). Additionally, we extended the GSK-3β studies into the field of epithelial to mesenchymal transition (EMT) research. Specifically we focused on understanding how GSK-3β in conjunction with the hypoxia inducible factor (HIF) proteins can influence the persistence of EMT and the production of vascular endothelial growth factor (VEGF). Collectively, these studies demonstrate important roles in lens epithelial cell mitoprotection for GSK-3β and ERK1/2; and, demonstrate a pivotal role for HIF-1α in the persistence of EMT under hypoxic conditions. Overall, the work described herein has provided invaluable information and understanding in the field of mitoprotection research as well as EMT research.