Recent developments in canine Cushing’s syndrome Nieuwe ontwikkelingen met betrekking tot het syndroom van Cushing bij de hond (met een samenvatting in het Nederlands) Novejša dognanja o Cushingovem sindromu pri psu (z izvlečkom v slovenščini) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 22 juni 2010 des middags te 12.45 uur door Sara Galac geboren op 16 januari 1968 te Slovenj Gradec, Slovenië Promotor: Prof.dr. J. Rothuizen Co-promotoren: Dr. H.S. Kooistra Dr.ir. J.A. Mol The studies described in this thesis were conducted at and financially supported by the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Publication of this thesis was made possible by generous support of: AUV Dierenartsencoöperatie Boehringer Ingelheim B.V. Dechra Veterinary Products Eurovet Animal Health J.E. Jurriaanse stichting Novartis Animal Health Royal Canin Virbac Ontwerp en grafische begeleiding: Kees van Veenendaal, Haarlem Omslagillustratie: Ruby de Goede, Heemstede Druk: Drukkerij de Bij, Amsterdam CIP-DATA KONINKLIJKE BIBLIOTHEEK, DEN HAAG Galac, Sara Recent developments in canine Cushing`s syndrome Sara Galac, Utrecht Universiteit Utrecht, Faculteit Diergeneeskunde Thesis Universiteit Utrecht. – With ref. – With summary in Dutch and Slovene. ISBN 978-90-393-5347-9 Subject headings: hypercortisolism, adrenocortical tumor, dogs Contents Chapter 1 Aims and scope of the thesis 7 Chapter 2 General introduction 11 Part 1: Pathogenesis of adrenocortical tumors in dogs with hypercortisolism Chapter 3 Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide and vasopressin in normal adrenals and cortisol- secreting adrenocortical tumors in dogs 45 Chapter 4 Expression of the adrenocorticotropic hormone receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors 69 Part 2: Diagnosis and treatment of Cushing`s syndrome in dogs Chapter 5 Urinary corticoid:creatinine ratios in the differentiation between pituitary-dependent hypercortisolism and hypercortisolism due to adrenocortical tumor in the dog 89 Chapter 6 Effects of trilostane treatment on the pituitary-adrenocortical and renin-aldosterone axis in dogs with pituitary-dependent hypercortisolism 99 Chapter 7 Urinary corticoid:creatinine ratios in dogs with pituitary- dependent hypercortisolism during trilostane treatment 115 Part 3: New forms of hypercortisolism in the dog Chapter 8 Hypercortisolism in a dog due to ectopic secretion of adreno- corticotropic hormone 131 Chapter 9 Adrenocorticotropic hormone-independent hypercortisolism due to food-dependent hypercortisolemia in a dog 147 Chapter 10 Summarizing discussion and conclusions 161 Chapter 11 Samenvatting en conclusies 177 Chapter 12 Izvleček in zaključki 191 Acknowledgments/Dankwoord/Zahvala 204 Curriculum Vitae 210 List of publications 212 Recent developments in canine Cushing’s syndrome Chapter 1 Aims and scope of the thesis 7 Recent developments in canine Cushing’s syndrome 8 Hypercortisolism or Cushing`s syndrome is one of the most common endocrinopaties in dogs. In about 80-85% of cases, hypercortisolism in dogs arises from hypersecretion of adrenocorticotropic hormone (ACTH) by a pituitary corticotroph adenoma and is called ACTH- or pituitary-dependent hypercortisolism (PDH). In the remaining cases canine hypercortisolism is ACTH-independent and results from excessive secretion of glucocorticoids by a benign or malignant adrenocortical tumor (AT). In the general introduction of this thesis (Chapter 2) an overview is given of pituitary and adrenal morphology and their regulation. Furthermore, symptoms and signs, the diagnostic approach, and treatment options in dogs with hypercortisolism are descri- bed. In addition, less common forms of hypercortisolism in humans, such as ACTH- independent hypercortisolism due to aberrant expression of adrenocortical hormone receptors and ectopic ACTH secretion are introduced. The mechanisms by which cortisol is produced in ACTH-independent hypercortiso- lism were previously classified as autonomous. However, recent research in humans has revealed that steroidogenesis in some cortisol-producing adenomas is stimulated by aberrant expression of adrenocortical hormone receptors. The presence of either ectopic or overexpressed eutopic G-protein coupled hormone receptors regulates steroidogenesis by mimicking cellular events that are triggered normally by the ACTH receptor. This leads not only to hypercortisolemia, but most likely also to hyperplasia, proliferative advantage, and tumor development. To provide the answer whether aberrant adreno- cortical hormone receptors play a role in the pathogenesis of ATs in dogs with hyper- cortisolism, the mRNA expression of gastric-inhibitory polypeptide (GIP) receptor (GIPR), luteinizing hormone receptor (LHR), and vasopressin (V1a, V1b, and V2) receptors was studied by quantitative PCR (QPCR) and the protein expression and localization of these receptors were studied by immunohistochemistry (Chapter 3). Alterations in steroidogenesis due to upregulation of genes encoding for steroidogenic enzymes of the cortisol-pathway could also provide an explanation for hypercortisole- mia in ACTH-independent hypercortisolism. This supposition seems especially at- 1: Aims and scope of the thesis tractive because in dogs with cortisol-secreting ATs the plasma ACTH concentration is suppressed, which suggests that the steroidogenesis is not initiated by binding of ACTH to its receptor (ACTH-R), like under physiological circumstances. In an at- tempt to obtain more information about the steroidogenesis in dogs with ACTH- independent hypercortisolism due to AT, the expression of mRNA encoding steroido- genic enzymes needed for cortisol biosynthesis, steroidogenic acute regulatory protein (StAR), and ACTH-R were studied (Chapter 4). Differentiation between PDH and adrenal hypercortisolism is mandatory to allow selection of the best treatment. The intravenous high-dose dexamethasone suppression 9 test (HDDST) is often used to differentiate between these two forms of the disease. In the intravenous HDDST a decrease of more than 50% from baseline plasma cortisol concentration confirms pituitary dependency, whereas a decrease of less than 50% points to dexamethasone resistance. Whether this 50% criterion for dexamethasone resistance can also be adopted when the urinary corticoid:creatinine ratio (UCCR) is used for the diagnosis of hypercortisolism was explored in Chapter 5. Trilostane has become the medical treatment of choice for canine PDH. Trilostane is a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (HSD3B), an essential enzyme system for the synthesis of cortisol, aldosterone (ALD), and androstenedione. In dogs with PDH, trilostane has the potential to induce a substantial decrease in basal and ACTH-stimulated plasma cortisol concentrations. Based on the mechanism of action, trilostane treatment is expected to affect both the pituitary-adrenocortical axis and the renin-ALD system. To test this assumption, measurement of the plasma concentrations of ACTH and cortisol and their ratio was used to assess the pituitary- adrenocortical axis and measurement of the plasma ALD concentration and plasma renin activity and their ratio was performed to assess the renin-ALD system in dogs with PDH receiving the optimal trilostane dose and in few dogs that received a trilo- stane overdose (Chapter 6). The effectiveness of trilostane therapy is judged by resolution of the clinical signs associated with glucocorticoid excess and results of an ACTH stimulation test. The aim of performing the ACTH stimulation test in dogs on trilostane therapy is to eva- luate whether sufficient adrenocortical reserve is present at the time of maximal effect of trilostane, which is 2-3 hours after its administration. The main disadvantages of this approach are that it only provides information about suppression of cortisol production during a short interval and that the post-ACTH cortisol concentration thought to indicate optimal dosage of trilostane is still arbitrary. Measuring the UCCR might be a more appropriate indicator of the therapeutic efficacy of trilostane, since the UCCR represents an integrated measure of glucocorticoid production. The results 1: Aims and scope of the thesis of UCCR determination before and during trilostane therapy in dogs with PDH are presented in Chapter 7. Ectopic ACTH secretion syndrome accounts for about 15% of cases of ACTH- dependent hypercortisolism in humans and comprises a spectrum of tumors from undetectable isolated lesions to wide-spread metastatic and aggressive malignancies. It is often associated with severe hypercortisolemia causing hypokalemia, diabetes mellitus, and general infections. Localizing the source of ectopic ACTH hypersecretion in humans is essential to stage the disease and to adopt optimal treatment modalities. 10 In Chapter 8 the first documented case of a dog with hypercortisolism due to ectopic ACTH secretion is presented. Bilateral adrenocortical hyperplasia is a form of ACTH-independent hypercortisolism in humans as a result of aberrant expression of adrenocortical hormone receptors. Adrenal hormonal secretion in these cases is induced by the binding of the specific ligand to these