Renin-Angiotensin-Aldosterone System (RAAS) and Hypothalamic-Pituitary- Adrenal Axis (HPAA) in Critically Ill Foals THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Katarzyna Agnieszka Dembek Graduate Program in Veterinary Clinical Sciences The Ohio State University 2012 Master's Examination Committee: Associate Professor Ramiro Toribio; Advisor Professor Catherine Kohn Assistant Professor Samuel Hurcombe Copyrighted by Katarzyna Agnieszka Dembek 2012 Abstract Sepsis is a major cause of morbidity and mortality in neonatal foals. Dysfunction of the hypothalamic-pituitary-adrenal axis (HPAA), manifested as relative adrenal insufficiency (RAI), has been associated with sepsis in newborn foals. Information on the renin- angiotensin-aldosterone system (RAAS) is minimal in healthy or sick foals. The HPAA and RAAS are interactive systems, and a relationship between RAAS activation and RAI is well documented in critically ill children, but limited information exists in septic foals. We hypothesized that in critically ill septic newborn foals the RAAS and HPAA will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. For this project, 167 (study 1) and 182 (study 2) sick and healthy foals of less than 7 days of age were included. Blood samples were collected on admission from septic (sepsis score >12), sick non-septic (SNS), and healthy foals. Blood concentrations of cortisol, aldosterone, angiotensin-II (ANG-II), corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH) and plasma renin activity were determined by immunoassays. Aldosterone, ANG-II, ACTH and cortisol concentrations were higher in septic compared to healthy foals (P<0.05). AVP was higher and CRH was lower in septic than healthy iii foals. Septic foals had higher ACTH/aldosterone and ACTH/cortisol ratios than healthy foals (P<0.05). No differences in plasma renin activity were found. Sepsis activates RAAS and HPAA in newborn foals. RAAS activation in critically ill foals is characterized by increased aldosterone and ANG-II concentrations. Low CRH concentrations in septic foals were an unexpected finding. We propose that AVP (not CRH) is the main ACTH releasing-hormone in critically ill foals. Septic foals demonstrated adrenocortical exhaustion with high ACTH/cortisol and ACTH/aldosterone ratios, indicating that RAI is not restricted to the zona fasciculata, but also involves the zona glomerulosa. iv Dedicated to my parents for their love, endless support and encouragement v Acknowledgments I would like to acknowledge my adviser, Dr. Ramiro Toribio for his intellectual support and assistance in my research realization. Special thanks go to Dr. Catherine Kohn, I am endlessly grateful for her mentorship in the clinical setting of my residency. I am thankful for the support and advice from Dr. Samuel Hurcombe for his clinical mentorship and advice. I am also indebted to Steven Naber and Sasha Bai for their statistical expertise. I would like to thank all of the clinicians and technical staff at Hagyard Equine Medical Center and Rood and Riddle Equine Hospital in Lexington, Kentucky for their dedication and support of this project. I also found myself lucky to have Kate Onasch, Brandy Marlow, Krista Hernon and Eason Hildreth III to help me in the lab and with data retrieval. Without the support from the aforementioned people, this project would have been very difficult to complete. vi Vita since 07/09 Residency and Graduate Teaching and Research Assistant, Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, USA 01/07 - 05/09 Associate, Al- Khalediah Equine Hospital , Saudi Arabia 01/06 - 12/07 Internship, Lisadell Equine Hospital, Ireland 06/05-12/05 Internship, Equine Clinic, Warsaw University of Life Sciences, Poland 09/99 - 05/05 School of Veterinary Medicine, Warsaw University of Life Sciences, Poland Fields of Study Major Field: Veterinary Clinical Sciences vii Table of Contents Abstract .............................................................................................................................. iii Acknowledgments.............................................................................................................. vi Fields of Study .................................................................................................................. vii Table of Contents ............................................................................................................. viii List of Tables ..................................................................................................................... ix Chapter 1: Introduction and Literature Review .................................................................. 1 2.1 Materials and Methods ................................................................................................ 12 2.2 Results ......................................................................................................................... 15 2.3 Discussion ................................................................................................................... 19 Chapter 3. Hypothalamic-pituitary-adrenal axis in hospitalized foals.............................. 32 3.1 Materials and Methods ................................................................................................ 32 3.2. Results ........................................................................................................................ 35 3.3 Discussion ................................................................................................................... 37 References ......................................................................................................................... 46 viii List of Tables Table 2.1 Blood hormone and electrolyte concentrations, and hormone ratios in neonatal foals ................................................................................................................................... 22 Table 2.2 Blood hormone concentrations and hormone ratios in surviving and nonsurviving septic foals . ................................................................................................ 24 Table 2.3 Hormone univariate analysis for survival among neonatal foals ..................... 25 Table 2.4 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in septic foals……………………………… 29 Table 2.5 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in SNS foals. ........................................................ 30 Table 2.6 Correlations (ρ) between hormones, serum electrolytes, IgG, creatinine and BUN concentrations, and sepsis score in hospitalized foals ............................................. 31 Table 3.1Blood hormone concentrations and hormone ratios in neonatal foals ............... 41 ix Table 3.2 Blood hormone concentrations and hormone ratios in surviving and non- surviving septic foals (values expressed as median and range) ........................................ 42 Table 3.3 Hormone univariate analysis for survival among neonatal foals ...................... 43 Table 3.4 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals. .............................................................................. 44 Table 3.5 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in SNS foals. ................................................................................ 44 Table 3.6 Correlations (ρ) between hormones, IgG, sepsis score, total protein, and glucose concentrations in septic foals. .............................................................................. 44 x Chapter 1: Introduction and Literature Review 1.1 Sepsis in neonatal foals Sepsis/septicemia, defined as the presence of bacteria or bacterial toxins in the bloodstream can lead to organ dysfunction and death in newborn foals.1-4 Sepsis is the main cause of foal mortality in the first week of life, resulting in major economical losses to the equine industry.1,1-9 Most of clinical signs of sepsis and septic shock are caused by a cascade of inflammatory mediators released in response to bacterial toxins. This cascade leads to the Systemic Inflammatory Response Syndrome (SIRS). When the initial protective response against sepsis is overwhelmed, the end result is Multiple Organ Dysfunction (MODS).1 SIRS is not restricted to bacterial insults but it may also be caused by viruses, fungi, protozoa, extensive trauma, hypoxia, ischemic disease and certain drugs (mostly anti-neoplastic). Any type of infection (bacterial, fungal or viral) can result in sepsis/SIRS, however, bacterial infections are the most frequent cause of severe sepsis in neonatal foals.1-4,6-9 The most common routes of infections in newborn foals include: respiratory tract, gastrointestinal tract, placenta, umbilical structures, and the integumentA number of pathogens are involved in pathogenesis of septicemia in 1 neonatal foals.3,4,9 Escherichia coli, Actinobacillus equuli, Salmonella spp. and Klebsiella pneumoniae are the most commonly isolated gram negative organisms, while Streptococcus spp., Staphylococcus aureus, and Clostridium spp. are the