Date of Award

8-1-2014

Degree Type

Thesis

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Robert T. Mallet

Second Advisor

Myoung-Gwi Ryou

Third Advisor

Rong Ma

Abstract

Introduction: 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 < 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

Comments

Hollrah, Roger A., Intravenous Pyruvate to Prevent Renal Injury Following Cardiac Arrest and Resuscitation. Master of Science, August 2014, 60pp., 25 figures, bibliography: 43 titles. Available online August 2015.