12-01-2011 - Recent developments in canine Cushing’s syndrome
Recent developments in canine Cushing’s syndrome
“Cortisol is the principal glucocorticoid released by the adrenals in dogs and cats. Thus endogenous glucocorticoid excess is essentially hypercortisolism. Prolonged exposure to inappropriate elevated plasma concentrations of free cortisol leads to symptoms and signs often referred to as Cushing’s syndrome, after Harvey Cushing, the neurosurgeon who in 1932 first described the syndrome in man. Identical symptoms and signs are elicited by exogenous glucocorticoids in long-term therapy”. [After: Rijnberk A. and Kooistra H. (eds.) “Clinical endocrinology of dogs and cats” 2e ed. Hannover: Schlütersche Verlagsgesellschaft mbH & Co, 2010, p. 111.]
Morphology and regulation of the canine pituitary gland
The pituitary gland or hypophysis (glandula pituitaria or hypophysis cerebri) is attached to the diencephalon on the ventral midline. Although small, it plays a major regulatory role in the entire endocrine system. The close anatomical relation of its glandular and nervous parts is symbolic of its function in interrelating the nervous and endocrine systems. So extensive are the influences of the hypophysis upon cells, tissues, and organs that it is often referred to as the “master gland” of the body. Its location as an appendage of the brain also points to its significance as the relay between the nervous and humoral mechanisms that jointly control multiple key body functions .
The mature pituitary gland consists of two major parts, the adenohypophysis (AH) and the neurohypophysis (NH). The canine hypophysis is suspended from the midline of the hypothalamus by a short cylindrical stalk. This stalk is the proximal portion of the NH and is called the pars proximalis NH or infundibulum. In most dogs the third ventricle continues as an invagination, the recessus NH, into the infundibulum. The pars proximalis NH is continuous with the distal enlargement, the pars distalis NH, which is the major portion of the neurohypophysis. The pars distalis NH is in direct contact with the inner part of the AH, the pars intermedia (PI), its name referring to its location between the two major parts of the hypophysis. The largest portion of the AH remains separated from the PI by the hypophyseal cleft and forms the distal portion of the AH, the pars distalis AH or anterior lobe (AL). The AH also extends as a cuff or collar around the pars proximalis NH to envelop part of the median eminence. This is the pars infundibularis AH  (Figure 1).
The pars distalis AH is populated by at least five highly differentiated types of endocrine cells, classified according to the tropic hormones they produce: somatotropic cells secreting growth hormone (GH), lactotropic cells secreting prolactin, thyrotropic cells secreting thyroid-stimulating hormone (TSH), gonadotropic cells secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and corticotropic cells synthesizing the precursor molecule pro-opiomelanocortin (POMC), which gives rise to adrenocorticotropic hormone (ACTH) .
Figure 1. Schematic illustration of an median sagittal section through the canine pituitary gland (With permission, Meij et al. 1997). Left is rostral, right is caudal. 1 = sphenoid bone, 2 = tuberculum sellae, 3 = dorsum sellae, 4 = pituitary fossa, 5 = pars distalis adenohypophysis, 6 = pars infundibularis adenohypophysis, 7 = transitional zone, 8 = hypophyseal cleft or cavity, 9 = pars intermedia adenohypophysis, 10 = third ventricle, 11 = hypothalamus (median eminence), 12 = pars proximalis neurohypophysis, 13 = pars distalis neurohypophysis.
POMC is also synthesized in cells of the PI, where two types of POMC-producing cells have been identified. One type is similar to the corticotroph cells of the AL in that it reacts with anti-ACTH in immunohistochemical staining. In the other type, ACTH is cleaved into ACTH1-14 (precursor of α-melanocyte stimulating hormone (α-MSH)) and corticotropin-like intermediate-lobe peptide (CLIP or ACTH18-39) (Figure 2). The PI is poorly vascularized and is directly innervated by predominantly dopaminergic nerve fibers from the hypothalamus. This direct neural control is mainly inhibitory in nature. Although there are high levels of bioactive ACTH in the canine PI, its main secretory product is α-MSH  .
Figure 2. Posttranslational cleavage of pro-opiomelanocortin (POMC) in the anterior lobe (AL) and pars intermedia (PI) of the pituitary gland. ACTH = adrenocorticotropic hormone, J PEPTIDE = joining peptide, β-LPH = β-lipoprotein, α-MSH = α-melanocyte-stimulating hormone, CLIP = corticotropin-like intermediate lobe peptide, β-END = β-endorphin.
The pars distalis NH consists of cells (pituicytes) and unmyelinated nerve fibers derived from neurosecretory neurons in the hypothalamus. These nerve fibers contain oxytocin or vasopressin in transit for secretion from the end of the axon into the systemic circulation. They are stored in secretory granules within the nerve terminals in the NH and are released by exocytosis into the bloodstream in response to appropriate stimuli . Hormone secretion from the pars distalis AH is regulated by the intimate contact between the hypothalamus and pituitary gland maintained via the hypophysial portal system. In the median eminence the hypothalamic releasing hormones (HRH) are secreted from the axon terminals of the hypothalamic neurons into fenestrated capillaries. Since the portal blood flow to the pituitary is not compartmentalized, the HRH gain access to all types of endocrine cells in the AL. Selectivity rests on the expression of specific receptors for HRH on the five different types of cells in the AL. However, the classic concept that hormone secretion from each cell type is regulated by a specific HRH has become modified by the finding of multiresponsive cells. These individual AH cells express multiple HRH receptors and are involved in critical endocrine events, such as lactation and adaptation to stress and low temperatures .
Hormone release from the AH is subject to a negative feedback (closed-loop) system. In a long-loop feedback, the hormones produced in the target endocrine organs, such as the thyroids, adrenals, and gonads, act on both the pituitary and the hypothalamus. In a short-loop feedback, some pituitary hormones, such as prolactin, regulate their own secretion by acting on the hypothalamus . Additionally, there is evidence of an ultra-short loop, by which the hormone acts within the hypophysis through autocrine and paracrine communication . Superimposed on these blood-borne regulatory mechanisms there are other signals, mediated by neurotransmitters and hypophysiotropic hormones, that represent the influence of environment, stress, and intrinsic rhythmicity .
Morphology of the canine adrenal gland
The adrenal gland (glandula suprarenalis) is located near the craniomedial border of the kidney. The topographic relation of the adrenal to the kidney in humans and other primates in the standing position led to use of the term suprarenal gland. The term glandula adrenalis, introduced in the sixth edition of the Nomina Anatomica  is more appropriate in dogs.
The adrenal gland is composed of a cortex and a medulla, two structurally and functionally different tissues having different developmental histories. The cortex is of mesodermal origin and is derived from the patch of celomic epithelium close to the gonadal fold. The medulla arises from neural crest cells, which migrate from their point of origin into the developing mesodermal mass . The adrenal capsule (capsula adrenalis) develops as a condensation of the mesenchyme at the periphery of the cortex. The outer portion, pars fibrosa, becomes a supportive fibrous stroma. The inner portion of the capsule at birth and in the young remains quite cellular and during this period is termed the pars cellulosa. Until the development of the adult cortex, this inner cellular layer can serve as the stem cell population for the generation of additional adrenal cortical parenchyma . The outermost zone of the adrenal cortex is the zona arcuata or zona glomerulosa (ZG). It is composed of cells arranged in arches and nestled in a stromal template provided by the inner surface of the capsule. The next cortical zone, the zona fasciculata (ZF), is the thickest. Its narrow outer surface is called zona intermedia corticalis. This small region comprises less than 5% of the total cortex and functions as a blastemic region for replacement cells of the adult cortex  . The major part of ZF is composed of anastomosing plates or muralia of cells that radiate toward the periphery . The cells of the outer one-third of this zone contain more lipid and are somewhat larger than those of the inner two-thirds. The innermost cortical layer is applied to all surfaces of the undulating contour of the medulla. The parenchyma of this inner zone is disposed in a relatively random and loose network and is termed the zona reticularis (ZR). The medulla, the central core of the gland, is separated from the cortex by a delicate network of reticular and loose collagenous connecting tissues, the septum corticomedullae (Figure 3).
Figure 3. Histological section of the adrenal gland of a healthy dog stained with hematoxyline and eosin. A = medulla, B = zona reticularis (ZR), C = zona fasciculata (ZF), D = zona glomerulosa (ZG), E = capsule (C).
The adrenal cortex is a major steroid-producing organ. The ZG of the canine adrenal cortex secretes mineralocorticoids (primary aldosterone, ALD) and is deficient in 17α-hydroxylase activity (CYP17), which renders it incapable of synthesizing cortisol or androgens. In contrast, only cells in the ZG contain the enzymes necessary to synthesize ALD. The ZF and ZR function as a unit, secreting glucocorticoids (mainly cortisol) and small amounts of sex steroids (estrogens, progestins, and androgens). The adrenal medulla secretes catecholamines (adrenaline and noradrenaline) .
Regulation of adrenal gland steroid secretion
The secretion of glucocorticoids is under the control of the hypothalamic-pituitary-adrenocortical axis (HPA), which represents a complex set of direct influences and feedback interactions between the hypothalamus, the pituitary gland, and the adrenal gland. The main pituitary hormone regulating adrenal steroid secretion is ACTH. ACTH is synthesized in both the AL and the PI from the well-characterized precursor molecule, POMC. The PI is directly innervated by predominantly dopaminergic nerve fibers from the hypothalamus; this direct neural control is largely a tonic (dopaminergic) inhibitory influence. The AL is regulated by the hypothalamus and central nervous system via neurotransmitters that cause the release of hypophysiotropic hormones, such as corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP), into capillaries of the hypothalamic-hypophyseal portal system. The AVP in portal blood is derived primarily from CRH-containing parvocellular neurons that originate in the paraventricular nucleus and project to the median eminence, thereby being fully separated from the vasopressin involved in water homeostasis .
In the neuroendocrine control of ACTH secretion the following mechanisms can be distinguished: episodic secretion, response to stress, feedback inhibition of cortisol, and immunological factors . Like the other hormones of the pituitary, ACTH is secreted in a pulsatile fashion (as consequently is cortisol). Central nervous system events regulate both the number and magnitude of ACTH bursts, ranging in the dog from six to twelve per 24h period 19]. There seems to be no circadian rhythm in cortisol secretion in dogs   . Stress responses originate in the central nervous system and increase the release of CRH and AVP. ACTH and cortisol are secreted within minutes following the onset of stress  . Using urinary cortisol as an integrated measure of cortisol production, stress such as exposure to veterinary procedures is reflected in elevated urinary corticoid:creatinine ratios (UCCR) .
Feedback inhibition is another major regulator of ACTH and thus cortisol secretion. The inhibitory action of glucocorticoids is exerted at multiple target sites, of which two have been unequivocally identified: neurons in the hypothalamus that produce CRH and AVP, and corticotropic cells in the AL. The feedback actions of glucocorticoids are exerted through at least two structurally different receptor molecules: a mineralocorticoid-preferring receptor (MR) and a glucocorticoid-preferring receptor (GR). The MR has a 20-fold higher affinity for cortisol than does the GR. There is evidence that inhibition of basal ACTH secretion by glucocorticoids is mediated via occupancy of the MR. The dog brain and pituitary contain very high levels of MR, the highest being in the septohippocampal complex and the pituitary AL . The GR is more evenly distributed in the brain, but the level in the AL is about twice as high as elsewhere, being mainly involved in the feedback effect of glucocorticoids released as a result of stress-induced ACTH secretion.
Challenges to the immune system by infection invariably activate the HPA. This response is mediated by proinflammatory cytokines, a group of polypeptides released from colonies of activated immune cells. Among the several cytokines, particularly interleukin (IL-6) activates the HPA . It is released from activated macrophages in the periphery and is also produced in the brain . The regulatory actions of the cytokines are exerted predominantly at the level of the hypothalamus, where CRH is the major mediator of the hypothalamic response. Cytokine-mediated activation of the HPA is also subject to feedback regulation by glucocorticoids, which not only impair the hypothalamic response to cytokine activation but also block cytokine production and inhibit macrophages. Thus there is bidirectional communication between the neuroendocrine system and the immune system .
ALD is the most potent mineralocorticoid and the three main mediators of its release are the renin-angiotensin system (RAS), potassium, and ACTH . In turn, the most sensitive mediator of renin release is renal perfusion pressure, changes in which are sensed by myotransducers in the wall of the afferent arteriole. The resulting signals are transmitted to the juxtaglomerular apparatus to modify the level of renin release and, therefore, ALD secretion . Adjacent to the afferent arteriole, cells of the macula densa in the distal tubules “sense” the composition of the tubular fluid. Signals from these cells may be responsible for a rapid change in circulating renin levels . Among the factors inhibiting renin release, angiotensin II and potassium play the most important roles. Angiotensin II directly suppresses renin release through a short feedback loop, independent of changes in blood pressure, ALD secretion, or renal blood flow. In humans, the adrenergic nervous system also modifies the release of renin, particularly in response to upright posture, with α and β stimulation having opposite effects ).
Potassium acts directly on the ZG cells by activating the conversion of cholesterol to pregnenolone . It suppresses renal renin release by directly inhibiting the release of renin-containing granules. Furthermore, potassium is a very potent stimulus for ALD secretion and small increases in plasma potassium concentration produce an immediate and significant increase in plasma ALD concentration .
ACTH is a potent acute ALD secretagogue but in both humans and experimental animals, the effect of ACTH infusion is short-lived unless it is pulsatile . During continuous ACTH stimulation, glomerulosa cell function appears to convert to fasciculata cell function and thus long-term ACTH stimulation paradoxically decreases ALD secretion.
An interesting feature of ALD secretion is the apparent likelihood that it is produced in other tissues. Although this is still controversial, studies have suggested that ALD production occurs in tissues such as the vasculature and the heart, but under the same control as ALD production by the adrenal gland cortex .
Adrenal gland steroid biosynthesis
Steroid hormones cannot be stored in steroidogenic cells but are secreted immediately after biosynthesis. Cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, and ALD are derived almost exclusively from adrenal secretion, whereas most other steroids are derived from a combination of adrenal and gonadal sources .
In the adrenocortical cell, glucocorticoid biosynthesis is initiated by binding of ACTH to its receptor (ACTH-R), which is a seven-transmembrane domain receptor belonging to a G-protein-coupled receptor subfamily. ACTH-R is a member of the melanocortin receptor subfamily, together with several other melanotropin receptors  . These receptors are characterized by short NH2-terminal extracellular domains, short intracellular COOH-terminal domains, short fourth and fifth transmembrane-spanning domains, and a small hydrophobic second extracellular loop . Until now, ACTH-R is the shortest known G-protein-coupled receptor, consisting of 297 amino acid residues with a predicted molecular mass of 33 kDa. It is mainly expressed in the adrenal cortex but has also been identified in human and canine skin   and rodent adipocytes . There is no evidence of ACTH-R expression in other tissues, which implies the existence of an effective mechanism to restrict expression of this gene. The regulation of the ACTH-R gene is unique in that it is upregulated by its own ligand in a time- and dose-dependent manner  . This unusual positive feedback loop might be an important adaptive process directed towards optimizing adrenal responsiveness to ACTH in the context of physiological stress and the maintenance of metabolic homeostasis in which the adrenals play a pivotal role .
All steroid hormones are derived from cholesterol. The adrenocortical cells can synthesize cholesterol de novo from acetate, mobilize intracellular cholesterol ester pools, or import lipoprotein cholesterol from plasma. About 80% of the cholesterol is usually provided by circulating plasma lipoproteins . Adrenal tissue in vitro utilizes low-density-lipoprotein (LDL) cholesterol via a specific receptor-mediated pathway . Specific cell surface receptors for LDL bind it and internalize it by receptor-mediated endocytosis. Under normal conditions, cholesterol de novo synthesis from acetyl coenzyme A represents about 20% of steroidogenic capacity. The amount of free intracellular cholesterol available for adrenal steroidogenesis is metabolically regulated, and negative feedback is exerted via the LDL pathway to control the amount of free intracellular cholesterol in adrenocortical cells. LDL uptake reduces the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol biosynthesis. ACTH increases the number of LDL receptors on the cell surface and the cholesterol esterase activity that liberates cholesterol from esters delivered by LDL or stored in lipid droplets, and thus the amount of free intracellular cholesterol . ACTH does not stimulate HMG-CoA reductase activity or alter the ability of LDL to suppress it.
When stimulation occurs by the binding of ACTH to its receptor, a Gs protein activates adenyl cyclase, causing the formation of cAMP. Protein kinase A is then activated and phosphorylates cholesterol ester hydrolase, thereby increasing its activity. Consequently, more free cholesterol is formed from stored cholesterol and there is increased uptake from plasma lipoproteins, as well as increased cholesterol synthesis in the adrenals. The acute response to a steroidogenic stimulus is mediated by steroidogenic acute regulatory protein (StAR). This mitochondrial phosphoprotein enhances cholesterol transport from the outer to the inner mitochondrial membrane  . The conversion of cholesterol to pregnenolone is the first and rate-limiting step in the cortisol steroidogenic pathway. It occurs in the mitochondria and involves the action of the cholesterol-side-chain cleavage enzyme (CYP11A). The newly synthesized pregnenolone is returned to the cytosolic compartment, where in a series of steps, microsomal enzymes convert it to 11-deoxycortisol.
The first step is conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase-type 2 (HSD3B), the only non-cytochrome P450 (CYP) enzyme in adrenal steroidogenesis. The following step is catalyzed by CYP17, which has both 17α-hydroxylase activity and 17,20-lyase activity . This dual function allows the enzyme to direct steroid precursors along several different pathways: 17α-hydroxylated substrates with the side chain are glucocorticoid precursors (17-OH progesterone and 17-OH pregnenolone), whereas both 17α-hydroxylase and 17,20-lyase direct substrate toward androgen and estrogen synthesis (dehydroepiandrosterone and androstenedione). Both progesterone and its 17α-hydroxylated derivate undergo 21-hydroxylation by a single 21-hydroxylase (CYP21) in the smooth endoplasmic reticulum. The last step in cortisol biosynthesis is 11b-hydroxylation of 11-deoxycortisol, catalyzed by 11b-hydroxylase-type 1 (CYP11B1). For this step, 11-deoxycortisol is transferred from the smooth endoplasmic reticulum back to the mitochondria  (Figure 4).
Figure 4. Major pathways of adrenocortical steroid biosynthesis. StAR = steroidogenic acute regulatory protein, CYP11A = cholesterol side-chain cleavage enzyme, HSD3B = 3b-hydroxysteroid dehydrogenase, CYP17 = 17a-hydroxylase, CYP21 = 21-hydroxylase, CYP11B1 = 11b-hydroxylase type 1, 17βHSD = 17β-hydroxysteroid dehydrogenase. Cellular location of enzymes (mitochondria or smooth endoplasmic reticulum) is depicted.
The secretion of ALD is restricted to the ZG. The biosynthetic pathway of ALD is initially similar to that of cortisol except that 17-hydroxylation does not occur, because of the lack of CYP17. In ALD synthesis progesterone is metabolized in the endoplasmic reticulum to 11-deoxycorticosterone. This is transferred to the mitochondria where the zone-specific 11β-hydroxysteroid dehydrogenase-type 2 (CYP11B2 or ALD synthase) converts it to corticosterone and progressively oxidizes it to ALD 
Spontaneous hypercortisolism is characterized by physical and biochemical changes resulting from chronic exposure to elevated circulating glucocorticoid concentrations. This disorder is often called Cushing’s syndrome, after Harvey Cushing, the neurosurgeon who first described the syndrome in humans in 1932.
About 80-85% of cases of hypercortisolism in dogs are ACTH-dependent, most often the result of excessive secretion of ACTH by a pituitary corticotroph adenoma. In the remaining cases canine hypercortisolism is ACTH-independent, the result of excessive secretion of glucocorticoids by benign or malignant adrenocortical tumors . In humans, hypercortisolism may also result from ectopic secretion of either ACTH or CRH . In addition, ACTH-independent hypercortisolism in humans may be due to bilateral macronodular adrenal hyperplasia and primary pigmented nodular adrenocortical disease .
Pituitary-dependent hypercortisolism (PDH) is one of the most common endocrine disorders in dogs. Exact incidence figures are lacking because there is no formal registration system, but some epidemiological studies have estimated the incidence to be 1 to 2 cases/1,000 dogs/year . In humans, PDH is a rare disorder with an estimated incidence of 1.2–2.4 new cases/million/year  . The pituitary lesions producing excess ACTH range from small nests of hyperplastic corticotrophs to adenomas and large pituitary tumors . The tumor may originate from the AL or the PI, both of which contain cells that can synthesize POMC, albeit with different posttranslational processing. In about 20-25% of cases there is a tumor in the PI. This is of clinical interest not only because tumors of the PI tend to be larger than those of the AL  , but also because of the difference in hypothalamic control of hormone synthesis in the two lobes , which is of importance in diagnosis
Hypercortisolism due to ectopic ACTH secretion
The ectopic ACTH syndrome accounts for about 15% of cases of ACTH-dependent hypercortisolism in humans. The majority of the ectopic tumors that secrete ACTH originate from cells of the diffuse neuroendocrine system and are highly malignant. They include small lung carcinomas, thymic and bronchial carcinoids, pancreatic islet cell tumors, medullary carcinomas of the thyroid, and pheochromocytomas  . The rapidly developing physical changes are usually associated with extremely high plasma concentrations of ACTH and cortisol and often severe hypokalemia. In humans circulating levels of cortisol are usually higher in the ectopic ACTH syndrome than in other forms of hypercortisolism. The high cortisol concentration saturates the renal protective tubular enzyme CYP11B1. This allows cortisol access to type I MR, resulting in cortisol-induced mineralocorticoid effects such as arterial hypertension and hypokalemia .
Hypercortisolism due to the ectopic ACTH syndrome is often difficult to distinguish from PDH. Tumors causing the ectopic ACTH syndrome are frequently small and difficult to visualize  . In addition, incidental nonsecreting pituitary tumors occur in up to 10% of the human populat ion, giving a false-positive finding by pituitary imaging. On the other hand, pituitary imaging may fail to demonstrate a microadenoma causing PDH . Consequently, the differentiation between PDH and hypercortisolism due to ectopic ACTH syndrome relies heavily on biochemical testing. Although circulating ACTH concentrations tend to be higher in the ectopic ACTH syndrome than in PDH, there is a large overlap . Dynamic noninvasive testing of the pituitary-adrenocortical axis is the most reliable means of differentiating the causes of ACTH-dependent hypercortisolism  . Surgical resection of the primary lesion results in complete remission in up to 80% of cases. It is therefore mandatory to localize the source of ectopic ACTH secretion in order to stage the disease and determine the best treatment. Modern cross-sectional imaging techniques can identify the majority of the ACTH-secreting lesions, either initially or at follow-up reassessment . However, in 10-20% of patients with ectopic ACTH syndrome the source of ACTH hypersecretion remains occult, in spite of extensive investigation and prolonged follow-up. In such cases, medical treatment is applied to control hypercortisolemia and the diagnostic imaging is repeated after some time .
Cortisol-secreting adrenocortical tumors
ACTH-independent hypercortisolism in dogs is usually caused by cortisol-secreting adrenocortical tumors (ATs). Most ATs are unilateral solitary lesions, the two glands being affected about equally. Bilateral tumors occur in about 10% of cases  . Histologically, ATs are classified as adenomas or carcinomas, although the distinction can be difficult. Microscopic examination of a seemingly benign tumor may reveal its expansion into blood vessels . While expansion of tumor tissue into blood vessels is generally considered to be a hallmark of malignancy, ATs may be an exception to this rule . In humans a scoring system which takes into account not only the results of histopathological examination but also the clinical picture and follow-up has been introduced to improve the reliability of the differential diagnosis of AT . Such a classification may also have to be introduced in veterinary medicine. In addition, reliable molecular and immunohistochemical markers have the potential to make assessment of adrenal malignancy much easier  . The most frequent modulation in human ATs is overexpression of the IGF-II gene, observed in 85% of carcinomas   and associated with paternal isodisomy at the 11p15 locus . Another remarkable feature of human adrenocortical carcinomas is low expression of the ACTH-R gene and loss of heterozygosity (LOH)  . One of the major characteristics of carcinomas is expansive growth, which is negatively correlated with steroid secretion . Furthermore, carcinomas have been found to be unable to carry steroidogenesis to completion, resulting in plasma patterns of steroid precursors typical of enzymatic blockage of cortisol synthesis with resulting hypersecretion of adrenal sex hormones . Mixed cortisol- and aldosterone-producing ATs have also been reported in dogs  . Simultaneous occurrence of an AT and a pheochromocytoma  or/and pituitary adenoma   has also been described in dogs.
Hypercortisolism due to aberrant expression of hormone receptors
In humans, ACTH-independent hypercortisolism can result from aberrant expression of adrenocortical receptors. Either ectopic or overexpressed eutopic adrenocortical hormone receptors can stimulate steroidogenesis and lead to the development of bilateral macronodular adrenal hyperplasia or an AT  . The ectopic hormone receptors that have been identified include the gastric-inhibitory polypeptide (GIP) receptor (GIPR), the renal vasopressin (V2) receptor (V2R), the pituitary-specific vasopressin (V1b) receptor (V1bR), the β-adrenergic receptor, and the angiotensin II receptor . The adrenocortical eutopic hormone receptors involved in hypercortisolism in humans include the vascular type vasopressin (V1a) receptor (V1aR), the LH receptor (LHR), and the serotonin 5-HT4 receptor. Most of these receptors belong to the superfamily of G protein-coupled receptors that have become aberrantly coupled to steroidogenesis . The presence of these receptors places adrenal cells under stimulation by a hormone that escapes the cortisol-mediated feedback system and leads to increased function and possibly to hyperplasia, proliferative advantage, and tumor development .
In veterinary medicine, LH-dependent hypercortisolism has been described in a pet ferret with a unilateral AT , and the presence of vasopressin receptors on ATs has been postulated by the finding of non-ACTH-mediated cortisol responses to systemic lysine vasopressin administration in dogs with ACTH-independent hypercortisolism .
Clinical manifestations of hypercortisolism in dogs
Spontaneous hypercortisolism is a disease of middle-aged and older dogs, although very rarely it has occurred as early as 1 year of age. There is no gender predilection. It occurs in all dog breeds, with a slight predilection for small breeds such as dachshunds and miniature poodles. Many of the clinical signs can be related to the biochemical effects of glucocorticoids, namely, gluconeogenesis and lipogenesis at the expense of protein. In dogs, the cardinal physical features are centripetal obesity and atrophy of muscles and skin. Polyuria and polyphagia are also dominating features. The polyuria is known to be due to impaired osmoregulation of vasopressin release and interference by the glucocorticoid excess with the action of vasopressin in the kidney. Abdominal palpation may reveal hepatomegaly .
Increased plasma alkaline phosphatase (AP) activity is a frequent finding in dogs with hypercortisolism. This is mainly due to the induction of an isoenzyme, which, having greater stability at 65 °C than other AP isoenzymes, is easily measured by a routine laboratory procedure . In about 50% of dogs with hypercortisolism, plasma thyroxine (T4) concentration is decreased as a consequence of altered transport, distribution, and metabolism of T4 rather than hyposecretion. Clinical changes associated with the pituitary origin of the disease are only observed when the pituitary tumor becomes large enough to cause neurological symptoms. These are often vague, consisting of lethargy, inappetence, and mental dullness . Clinical findings linked exclusively to an AT may be related to the adrenal mass and/or caused by metastases or nonspecific features of malignancy such as weight loss and anorexia. A palpable abdominal mass, obstruction of the caudal vena cava by tumor thrombi , or hemoperitoneum secondary to rupture of the tumor are rare consequences of an AT  .
Diagnosis of hypercortisolism
The biochemical diagnosis of hypercortisolism depends on the demonstration of two characteristics: (1) increased production of cortisol and (2) decreased sensitivity to glucocorticoid feedback . A single measurement of plasma cortisol concentration has little diagnostic value because the pulsatile secretion of ACTH results in a varying plasma cortisol concentration that may at times be within the reference range. There are two ways to overcome this problem: (1) by testing the integrity of the feedback system, and (2) by measuring urinary corticoid excretion. The first approach, testing the sensitivity of the pituitary-adrenocortical system to suppression, was introduced in 1960 . He demonstrated that when given a potent glucocorticoid such as dexamethasone in a dose too small to contribute to glucocorticoid measurements, patients with Cushing’s syndrome could be separated from those with normal adrenocortical function. This discovery continues to be applied and is the basis of the low-dose dexamethasone suppression test (LDDST) used to diagnose hypercortisolism in dogs. In normal dogs, intravenous administration of 0.01 mg dexamethasone per kg body weight suppresses plasma cortisol concentration to 40 nmol/l or less at 8 hours after dexamethasone administration  . In dogs with hypercortisolism there is little or no suppression at 8 hours . The LDDST is a reliable screening test with a reported sensitivity of 85%  or even 100%  and a specificity of 73%  .
The disadvantage of the LDDST is that it is rather time consuming and invasive, requiring blood collection at least twice. Hence is has been largely replaced by measurement of urinary corticoids. Because urine accumulates in the bladder for several hours before collection, its corticoid concentration is an integrated measure of corticoid production over this interval, smoothing out the effects of short-term fluctuations in plasma cortisol concentration. The urinary corticoids (largely cortisol) are related to the creatinine concentration in the urine, resulting in the UCCR. This test requires little time (from the veterinarian or the owner), is not invasive (no blood collection), and has a high diagnostic accuracy. In addition, the test procedure has the advantage of combining a test for basal adrenocortical function and a dynamic test for differential diagnosis (see below). To avoid the influence of stress, urine for measurement of the UCCR is collected at home, with an interval of at least one day after the visit to the veterinary clinic. The owner collects a sample of the first morning urine on two consecutive days and the UCCRs in these two samples are averaged. In our laboratory the basal UCCR in healthy pet dogs varies from 0.8 to 8.3 x 10-6 (Van Vonderen et al. 1998). In dogs the predictive value of a positive test result is 0.88 and that of a negative test result is 0.98 . In some dogs there is considerable day-to-day variation in the UCCR, which in mild forms of hypercortisolism occasionally leads to UCCRs just within the reference range, whereas urine collection on other days might reveal one or two elevated UCCRs.
In dogs in which results of the UCCR and/or the iv-LDDST have been inconclusive or negative but in which there is still suspicion of hypercortisolism, an oral LDDST can be performed. The owner collects urine at 8.00 h (at home) for measurement of the basal UCCR and then administers 0.01 mg dexamethasone per kg body weight orally. The dog is walked at 12.00 h and 14.00 h to empty its bladder and then urine is collected at 16.00 h to measure the effect of the low dose of dexamethasone on the UCCR. In 7 healthy pet dogs the UCCR at 16.00 h was <1.0 x 10-6  and in dogs with mild PDH it was >1.0 x 10-6 . The ACTH stimulation test has also been used in the diagnosis of hypercortisolism in dogs. In principle it is a test of adrenocortical reserve capacity, used to diagnose primary and secondary adrenocortical insufficiency, and thus it can be used to diagnose iatrogenic hypercortisolism, which via feedback suppression results in secondary adrenocortical insufficiency. However, the ACTH stimulation test was used more often in the past as a primary test to diagnose hypercortisolism. An exaggerated response, i.e., a greater elevation of plasma cortisol concentration than in healthy dogs, occurs in about 85% of dogs with PDH compared to about 55% of dogs with hypercortisolism due to AT . The main advantages of the ACTH stimulation test are its simplicity and short duration. However, its diagnostic accuracy for hypercortisolism is less than that of the UCCR and the LDDST. Hence it is no longer recommended for this purpose .
Differentiation between PDH and hypercortisolism due to AT
Once the diagnosis of hypercortisolism has been confirmed, it is necessary to distinguish between the different forms of the disease. Despite decreased sensitivity to suppression by glucocorticoids, which is a hallmark of hypercortisolism, in most dogs with PDH due to an adenoma in the AL, ACTH secretion can still be suppressed by a high-dose of dexamethasone . In contrast, PDH of PI origin is resistant to suppression by dexamethasone. The tonic dopaminergic inhibition, which is the principal neural control of the PI, suppresses the expression of glucocorticoid receptors in the PI. However, this is not an absolute difference from AL lesions, as pituitary lesions causing hypercortisolism do not always maintain the regulation characteristics of the lobe of origin. In PDH, the resistance to glucocorticoid feedback is significantly correlated with the size of the pituitary gland  .
The impaired sensitivity to glucocorticoid feedback in PDH due to an AL tumor can be demonstrated by performing an intravenous high-dose dexamethasone suppression test (HDDST). Blood for measurement of plasma cortisol concentration is collected immediately before and 3-4 h after intravenous administration of 0.1 mg dexamethasone per kg body weight. A decrease of more than 50% from the baseline value confirms PDH . When the suppression is less than 50%, the hypercortisolism may still be pituitary-dependent (due to either a PI tumor or a resistant AL tumor) or be due to an AT. Further differentiation requires measurements of plasma ACTH concentration. In animals with PDH, plasma ACTH concentration is not completely suppressed despite high plasma cortisol concentrations .
After the diagnosis of hypercortisolism has been established, diagnostic imaging of the pituitary gland and adrenals is of great assistance to determine the best treatment and objectively evaluate the prognosis. The pituitary can be visualized by computed tomography (CT) or nuclear magnetic resonance imaging (MRI). In dynamic contrast-enhanced CT, the absence of the pituitary flush indicates atrophy of the neurohypophysis and the adenohypophysis. Displacement of the pituitary flush in the early phase of the dynamic CT can be used to identify and localize microadenomas originating from the AL or PI in dogs  . The adrenal glands can also be visualized by CT or MRI  but because ultrasonography is less expensive, requires less time, and does not require anesthesia, it is often used first  . It can provide a good estimate of the size of an AT and may reveal its expansion into blood vessels and possible metastasis in the liver. If nodular structures are revealed in the liver, ultrasound-guided needle aspiration biopsy can be performed. Thoracic radiographs or a CT scan of the thorax should be made to determine whether there are metastases in the lungs.
The goal of treatment of hypercortisolism is to eliminate the clinical signs related to glucocorticoid excess. Depending on the cause, this may be achieved by transsphenoidal hypophysectomy, adrenalectomy, or medical treatment with o,p’-DDD (mitotane) or trilostane.
In hands of a skilled neurosurgeon transsphenoidal hypophysectomy is an effective treatment for canine PDH  . It is the only treatment that can eliminate the causative pituitary adenoma, but in the dog this requires complete removal of the pituitary. Following hypophysectomy, hormone replacement therapy consists of lifelong administration of cortisone acetate and thyroxine and temporary administration of desmopressin, a synthetic vasopressin analogue. The major complications are postoperative mortality, hypernatremia due to acute AVP deficiency, prolonged central diabetes insipidus, keratoconjunctivitis sicca, and residual or recurrent hypercortisolism . A recent study of the 10-year follow-up results in 150 dogs with PDH confirmed that it is effective, especially in the long term, with remission for up to seven years . However, the survival and disease-free periods decrease and the incidence of central diabetes insipidus increases with increasing pituitary size. Hence, transsphenoidal hypophysectomy can be expected to have the best outcome as the primary treatment in dogs with nonenlarged or only moderately enlarged pituitaries .
The treatment of choice for AT is adrenalectomy, because the successful removal of the affected adrenal gland eliminates the tumor and thus the clinical signs related to glucocorticoid excess, without the need for lifelong medication. Because of the atrophy of the cortex of the nontumorous contralateral adrenal gland, due to the longstanding glucocorticoid excess, glucocorticoid substitution is needed temporarily . Adrenalectomy can be performed in dogs via a ventral midline celiotomy, with a paracostal extension of the incision when needed, or via a paracostal approach   . Regardless of the surgical technique used, adrenalectomy is associated with a perioperative mortality of about 20%  . This can be explained in part by the relatively high risk of postoperative complications such as renal failure, pneumonia, pancreatitis, and pulmonary thromboembolism, but it also reflects the level of difficulty of the surgical procedure. In dogs with hypercortisolism the adrenals are usually poorly accessible due to central obesity and hepatomegaly. Furthermore, ATs are friable, lie in close proximity to the vena cava, and tend to invade blood vessels. As for hypophysectomy , the skill of the surgeon will affect the outcome.
In humans, laparoscopic adrenalectomy has been reported to have lower perioperative morbidity and mortality than open transabdominal surgery . It may also become the surgical procedure of choice in veterinary medicine , but as in humans, the size of the AT may be a limitation of this technique.
Selective  or nonselective  destruction of the adrenal cortex with o,p’-DDD (mitotane) has long been the medical treatment of choice for PDH in dogs. Some treatment schedules aim at selective destruction of the ZF and ZR, sparing the ZG. However, in 5-6% of dogs in which this is attempted, the ZG is also destroyed to such an extent that iatrogenic hypoadrenocorticism develops. On the other hand, in more than half of the cases in which selective destruction is the aim, there are one or more relapses of hypercortisolism during treatment. In order to avoid these complications, a treatment schedule with the aim of complete destruction of the adrenal cortices and substitution for the induced hypoadrenocorticism  was developed. This nonselective destruction has been reported to result in fewer recurrences than does selective destruction . However, o,p’-DDD is no longer regularly used for treatment of PDH, but rather for treatment of inoperable and/or metastasized ATs, with the aim of complete destruction of all adrenocortical cells, including metastases .
A few years ago, trilostane was reported to be a safe and effective alternative for o,p’-DDD in dogs with PDH  . Trilostane is a competitive inhibitor of HSD3B, an essential enzyme system for the synthesis of cortisol, ALD, and androstenedione . In dogs with PDH, treatment with trilostane has the potential to significantly decrease basal and ACTH-stimulated plasma cortisol concentration  . This results in loss of negative feedback and thus increased plasma ACTH concentration  . Consistent with its competitive inhibitory effect on HSD3B, trilostane also causes a slight decrease in plasma ALD concentration in dogs with PDH, although ALD usually remains within the reference range  . In humans, plasma renin activity rather than the plasma ALD concentration is considered to reflect mineralocorticoid deficiency . No detailed information is available on the effects of trilostane treatment on the RAS in the dog.
Trilostane is absorbed rapidly from the gastrointestinal tract. Administration with food significantly increases the rate and extent of absorption. There is marked variation in the optimal dose and to avoid adverse effects due to overdosage, treatment is started at a relatively low oral dose of 2 mg/kg body weight once daily. The effectiveness of trilostane therapy is judged by resolution of clinical signs associated with glucocorticoid excess and results of an ACTH stimulation test  . The aim of performing an ACTH stimulation test in a dog on trilostane therapy is to determine whether there is sufficient adrenocortical reserve at the time of maximal effect of trilostane, which is 2-3 hours after administration. The disadvantages of the test are that: (1) it only provides information about suppression of cortisol production during a short interval, (2) it is invasive, and (3) the post-ACTH cortisol concentration thought to indicate optimal dosage of trilostane is still arbitrary. Another way to evaluate trilostane therapy would seem to be measurement of the UCCR. This has been used to detect persisting cortisol secretion after hypophysectomy or nonselective destruction of the adrenal cortex by mitotane therapy  . Because the UCCR is an integrated measure of glucocorticoid production , it might be a more appropriate indicator of the therapeutic efficacy of trilostane than an ACTH stimulation test. Within about a week on an appropriate dose of trilostane, there is a clear reduction in water intake, urine output, and appetite, followed by improvement in the coat and skin, reduction of central obesity, and increased physical activity  .
Overdosage of trilostane results in cortisol deficiency and sometimes even mineralocorticoid deficiency  . In addition, necrosis, apoptosis, and hemorrhage in the ZF and ZR may cause life-threatening hypocortisolism . If this occurs, trilostane must be stopped immediately and corticosteroid substitution started. In most cases there is sufficient recovery of adrenocortical function within a few weeks and substitution can be stopped  .
The median survival time for dogs with PDH treated with trilostane once daily (662 days), is similar to that for selective adrenocorticolysis with o,p’-DDD (798 days) . The median survival time of dogs with PDH for treatment with trilostane twice daily (900 days) is comparable to that for the nonselective adrenocorticolysis with o,p’-DDD (720 days) . If there is metastasis of a functional AT or if neither adrenalectomy nor adrenocortical destruction with o,p’-DDD is an option, trilostane therapy can be used as a palliative treatment  .
Recent developments, conclusions       
-Hypersecretion of cortisol by canine ATs is not associated with overexpression of the genes encoding LHR, GIPR, V1aR, V1bR, or V2R.
-Ectopic expression of GIPR and V2R protein, and eutopic expression of LHR protein in tumorous ZF tissue, may play a role in the pathogenesis of canine cortisol-secreting ATs.
-Hypercortisolemia in dogs with cortisol-secreting ATs cannot be ascribed to upregulation of the genes encoding for steroidogenic enzymes.
-Significant downregulation of ACTH-R may contribute to the malignant character of cortisol-secreting carcinomas.
-The generally-accepted criterion of 50% suppression of plasma cortisol concentration by a high dose of dexamethasone in the differentiation between PDH and AT is also applicable to the UCCR.
-Trilostane treatment affects both the pituitary-adrenocortical axis and the renin-angiotensin-ALD system.
-The UCCR cannot be used as an alternative to the ACTH-stimulation test to determine the optimal dose of trilostane.
-The basal plasma ACTH concentration and the UCCR may both be used to detect dogs at risk for developing hypocortisolism during trilostane therapy.
-The ectopic ACTH secretion syndrome occurs in dogs and should be suspected in cases of severe and nonsuppressible hypercortisolemia.
-The differential diagnosis of canine ACTH-independent hypercortisolism should include FDH.
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