Abstract
Marked renal vascular changes, suggestive of hypertension, were present in adult western gray kangaroos (Macropus fuliginosus) from a single facility over a 14-year period. A subset of these kangaroos also had vague clinical nervous system deficits, including blindness. To characterize the vascular lesions, determine prevalence, and document other changes, case histories and archival tissue sections from 21 adult kangaroos (8 male, 13 female) that died or were euthanatized between 1994 and 2008 were reviewed. Relevant lesions included increased thickness of the renal arteriolar tunica media with smooth muscle hypertrophy and/or hyperplasia, accumulation of extracellular matrix within arterioles, increased vascular tortuosity, and varying degrees of juxtaglomerular hyperplasia. Renal tissue from two more severely affected animals was further examined by transmission electron microscopy, highlighting arteriolar endothelial cell hypertrophy and disruption of the medial architecture. Hypertrophy of arteries and arterioles in other organ systems was also present (3/21), including vessels in the brain and spinal cord of one animal with clinical neurologic signs. Four kangaroos had antemortem retinal detachment, a potential sequel of hypertension in humans and domestic mammals. The cause of these vascular lesions in this mob is uncertain. Lesions were not associated with an infectious disease process, age, underlying renal disease, or thyroid abnormalities. In the absence of other causes, hypertension was a differential. Further investigation into clinical significance and predisposing factors, such as genetics and diet, is warranted.
Introduction
At a single institution, over a 14-year period, multiple adult western gray kangaroos (Macropus fuliginosus) were presented for necropsy with a clinical history of vague neurologic signs and/or blindness. On microscopic examination, many of these animals had hypertrophic arteriolar and arterial walls, most consistently within the kidneys. Changes were similar to those documented in hypertensive humans and domestic mammals. The presence of these lesions suggest underlying hemodynamic changes are a contributing factor in the clinical signs and mortalities within the mob.
Hypertension is defined as persistently elevated peripheral vascular blood pressure in the face of normal cardiac output. Based on the cause, the disease may be classified as either essential or secondary. Essential (primary) hypertension is the most common type that affects humans. The disease is typically idiopathic, although multiple factors, such as genetics, stress, and nutrition, are thought to play a role in disease development. Secondary hypertension is the direct or indirect result of disease in another organ system, most commonly renal or endocrine. Secondary hypertension, the most common form diagnosed in domestic mammals, is usually attributed to renal disease. In dogs, hypertension has also been linked to functional pheochromocytomas, hyperadrenocorticism, hyperthyroidism, hypothyroidism, and diabetes mellitus. 1 In domestic cats, diabetes mellitus and hyperthyroidism are frequently associated conditions. 1,19,20,31 Hypertension may be further classified as benign or malignant. Malignant hypertension involves a rapid rise in blood pressure, often with a preexisting history of benign hypertensive disease. Malignant hypertension is associated with more severe, acute conditions, such as renal failure and retinal hemorrhage.
The purpose of this study was to review the clinical histories of these western gray kangaroos, the results of ancillary tests, and histologic and gross necropsy findings to further evaluate vascular changes and the pathogenesis of the lesions.
Materials and Methods
Animals
Medical records, archived tissues, and histologic sections from 21 adult (>1 year old) western gray kangaroos housed at a single facility between 1994 and 2008 were evaluated. Animals that had a complete necropsy performed after spontaneous death or euthanasia were included in the study. Ages at the time of death ranged from 3 to 17 years, with a mean age of 9.9 years and a median age of 10 years. There were 8 males and 13 females.
Histopathology
Complete tissue sets from all study kangaroos were fixed in 10% neutral buffered formalin, routinely processed for histology, sectioned at 5 µm, and stained with HE. In addition, a 3-µm-thick section of kidney and heart from two individuals with severe vascular hypertrophy (Nos. 7 and 3, respectively) were stained with periodic acid–Schiff (PAS) stain. Eyes from 13 kangaroos were available for evaluation.
To evaluate renal changes as objectively as possible, a grading scheme was developed to help describe varying degrees of arteriolar change. Because no such previously validated scale could be found in the published literature, for the purposes of this study glomeruli were assigned to 1 of 3 grades based on the severity of arteriolar microscopic changes. Glomeruli with a visible cross section of the afferent arteriole were graded based on afferent and efferent arteriolar and juxtaglomerular characteristics. Arterioles were defined as vessels with a muscular wall 100 µm or less in diameter. 16 Grade 0 arterioles were histologically within normal limits, having an afferent and efferent tunica media that consisted of a single layer of myocytes. Grade 1 arterioles were characterized by prominent endothelial cells with rounded nuclei and vesicular chromatin (hypertrophy), as well as 1–2 medial myocyte layers. Grade 2 arterioles were those with hypertrophied endothelial cells, 3–4 medial myocyte layers, and increased vascular tortuosity. Tortuosity was identified by a subjectively increased number of arteriolar profiles within a 500-µm radius of the glomerulus. Grade 3 arterioles had all of the above features in addition to 5 or more medial myocyte layers. The first 15 glomeruli from each kidney, randomly selected from all levels of the cortex and meeting the previously described standards, were evaluated from each kangaroo (30 per kangaroo). The overall grade for each kangaroo was calculated as an average of the 30 individual glomerular scores. To help determine whether age and severity of arteriolar changes were related, correlation analysis was applied by using the Pearson r-value and was calculated by using SAS 9.1 (Cary, NC).
Hyperplasia of the juxtaglomerular apparatus was assessed based on numbers of juxtaglomerular cells. Numbers of juxtaglomerular cells within grade 0 (histologically normal) glomeruli were considered to be within normal limits and ranged from 5 to 10 cells. Ten to 15 cells was considered to be mildly increased (grade 1), 15 to 25 cells were moderately increased (grade 2), and 25 or more cells were markedly increased (grade 3).
Transmission electron microscopy
Transmission electron microscopy was used to further evaluate kidneys from two of the more severely affected individuals (Nos. 2 and 7). Karnovsky-fixed tissues were postfixed in 2% osmium tetroxide, dehydrated, and embedded in epoxy resin. Sections were cut at 0.35 µm and stained with toluidine blue and basic fuchin for preliminary light microscopic review. Selected samples were then sectioned at 80–90 nm, stained with uranyl acetate and lead citrate, and examined with a Hitachi H600 transmission electron microscope (Hitachi America, Ltd., Brisbane, CA).
Results
Case details are summarized in Table 1. Clinical neurologic signs seen in 11 of 21 cases (52%) were acute, varied, and often vague. Five animals (Nos. 5, 7, 13, 17, and 18) were euthanatized because of worsening neurologic deficits.
Kangaroo clinical neurologic findings, retinal detachment and cause of death.
∗ N/A = tissues not available.
Sixteen individuals (76%) had an overall arteriolar grade of 1–3 and/or one or more grade 2 or 3 glomeruli (Table 2). Four had grade 3 changes (Nos. 1, 2, 3, and 4). Affected arterioles were randomly present at all levels of the renal cortex and were characterized by mild-to-marked concentric thickening of the tunica media, with increased numbers of smooth muscle cells (Figs. 1–3). Disruption of elastin fibers was occasionally visible on routine-stained sections. On a PAS-stained section from one of the grade 2 kidneys (No. 7), there were prominent intercellular accumulations of PAS-positive material within most arterioles. Additional histologic features included mild to moderately increased numbers of arteriolar profiles (tortuous). There was also mild medial hypertrophy of arcuate arteries in No. 2 and mild intimal to subintimal fibrosis of a renal artery of No. 3. Age at death did not correlate with average arteriolar grade (r-value of 0.257 and P > .05).
Kangaroo signalment, average and highest glomerular arteriolar grades, and degree of juxtaglomerular hyperplasia.
Kidney; western gray kangaroo No. 10. Grade 1 glomerulus. Afferent and efferent arterioles have prominent endothelial cells with vesiculate nuclei. Arteriolar walls have one to two myocyte layers. HE. Bar = 50 µm.
Kidney; western gray kangaroo No. 4. Grade 2 glomerulus. Arteriolar walls have 3–4 myocyte layers, and there is increased tortuosity of the vessels manifested as increased numbers of profiles. There are moderately increased numbers of juxtaglomerular cells. HE. Bar = 50 µm.
Kidney; western gray kangaroo No. 3. Grade 3 glomerulus. Arteriolar walls have 5 or more disorganized myocyte layers. Note the increased vascular tortuosity and small lumen diameters. HE. Bar = 50 µm.
Renal arterioles from No. 7 (Fig. 4) and No. 2 had similar ultrastructural features. Endothelial cells were elongate (“teardrop shaped”) and protruded into the lumen, and the luminal surfaces of the plasma membranes were slightly undulant. Within the tunica media, myocytes were shortened, and some had an indented nucleus. Medial intercellular junctions were thickened, with small accumulations of amorphous, electron-dense material that corresponded to the PAS-positive material in No. 7 and were interpreted as glycoprotein.
Renal arteriole; western gray kangaroo No. 2. Endothelial cells are rounded and protrude into the lumen. The luminal surface of the plasma membrane is undulant. Cell nuclei have moderate to large amounts of euchromatin. Elastin fibers are disrupted, and intercellular junction width is increased with small amounts of electron dense intercellular matrix. Transmission electron microscopy. Bar = 6 µm.
Kangaroos no. 2, 3, and 7 had severe thickening of arteriolar and arterial walls in tissues other than the kidneys, most often within the central nervous system (cerebrum and spinal cord), myocardium, gastrointestinal-tract walls, mesentery, spleen, thyroid gland, and occasionally lung. Extrarenal vascular changes were similar to those in the kidneys with markedly increased numbers of myocytes, associated reduction of lumen diameter, increased numbers of vascular profiles, multifocal disruption of elastin fibers, and accumulations of homogenous, PAS-positive intercellular matrix that was pale eosinophilic on HE-stained sections. Throughout the brain and spinal cord of No. 7, there were multiple clustered cross sections of hypertrophic arterioles, with only one accompanying vein. Within the spinal cord, some of these vessels were surrounded by small numbers of extravasated erythrocytes (acute hemorrhage).
Juxtaglomerular hyperplasia was present in most individuals, ranged from mild to severe and was generally multifocal. Hyperplasia manifested as increased numbers of disorganized, small epithelial cells within the mesangial tuft vascular pole (Figs. 2, 3). Other renal changes were infrequent, the most common being mild interstitial fibrosis and rare tubular mineralization (8/21). Only 2 individuals had other renal lesions considered severe enough to have caused renal compromise. No. 19 had tubular necrosis secondary to hyperthermia, and No. 9 had a severe bacterial pyelonephritis. In both cases, the lesions were acute, making an association with arteriolar changes unlikely.
In 4 individuals, there were bilateral or unilateral retinal changes that varied in severity between individuals and right and left eyes. All affected retinas had multifocal to diffuse atrophy, with variable loss of photoreceptors, loss of the outer plexiform and ganglion cell layers, and/or merging of inner and outer nuclear layers. There was multifocal to diffuse detachment of the outer retinal layers from the retinal pigmented epithelium. Based on apical rounding of pigmented epithelial cells at the sites of detachment (tombstoning), the separation was considered to be an antemortem change rather than artifact. The detached retinas were multifocally discontinuous with small accumulations of fibrous tissue and rounding (clubbing) of the fragmented margins. Arterioles adjacent to the optic nerves of No. 2 had mild-to-moderate accumulations of amorphous eosinophilic material within the walls (hyaline change); however, there were no appreciable vascular changes within the eyes of any individuals with retinal detachment. Two animals with retinal detachment had grade 3 renal arteriolar changes (Nos. 3 and 4); Nos. 12 and 13 also had retinal detachment, both animals had grade 2 lesions, and No. 13 had an overall grade of 1.36.
Concurrent lesions to explain the retinal detachment, neurologic signs or death were not present in 8 individuals (Nos. 3, 5, 7, 8, 12, 13, 14, and 18). Among these cases, one (No. 3) had grade 3 arteriolar lesions, and the others each had at least 25% grade 1 or grade 2 changes (data not shown). Thyroid adenomas were present in Nos. 3 and 4. The lesions were unilateral and bilateral, respectively.
Discussion
Histologic and ultrastructural arteriolar changes seen in the kidneys of many and multiple organ systems of several kangaroos in this study were morphologically consistent with hypertension. The kidneys, as blood-filtering organs, are readily responsive to blood-pressure alterations. The sequence of events leading to renal vascular microscopic changes in mammals is as follows; persistent afferent arteriolar reflex vasospasm and multifocal mural myocyte swelling progress to hyperplasia and hypertrophy and eventually fibrosis. During this process, arterioles become elongated and tortuous. Lumen diameter decreases because of both vasospasm and increases in wall thickness. 27 Ultrastructural features of thickened arterioles in the two examined kangaroo kidneys were consistent with prolonged constriction or vasospasm.
Although the lesions found in this study were morphologically compatible with hypertension, without antemortem blood pressure measurements, the diagnosis of hypertension cannot be made. A previous assessment of blood pressures among several species of kangaroo and wallaby found that cardiac, pulmonary, and peripheral pressures were similar between kangaroos and domestic dogs; however, western gray kangaroos were not specifically studied. 22 Blood-pressure evaluation of this mob is ongoing (Zachariah T, unpublished), and findings may shed some light on baseline values and aid in interpreting future fluctuations. It is important to be able to correlate high blood pressures with hypertensive disease in individual species, because some domestic canine breeds, for example, normally have higher baseline pressures. 25 Nonetheless, the lesions in the absence of other evident causes are suggestive of primary hypertension.
Persistent hypertension causes vascular-wall hypertrophy and hyperplasia via vascular-wall hemodynamic stress. Vessel-wall stretch receptors induce secretion of growth factors (such as transforming growth factor-β and platelet-derived growth factor), cytokines and angiotensin II, and production of extracellular matrix (collagen and elastin). 9,28 The addition of extracellular matrix and myocytes to the vascular wall disrupts the architecture, a feature evident in many of the affected kangaroo vessels. Disruptions of the normal architecture in conjunction with high luminal pressures ultimately weaken the structural integrity of the vessel wall. 3,16
Hypertrophy of endothelial cells is a common early hypertensive change, because endothelial cells are more immediately exposed to the bloodstream and, therefore, more sensitive to hemodynamic and cell-cell signaling. 3,9 In humans and rodents with hypertension, the severity of these lesions in the kidneys and other highly vascular areas, such as the lungs, correlate with increasing diastolic blood pressures. 3,5,15,24,26,29,32 Vascular lesions typically progress from intimal fibrosis to medial fibrosis to fibrinoid necrosis. In humans, fibrinoid necrosis is a prominent feature of malignant hypertension but has not been a consistent or common feature in hypertensive domestic dogs and cats. 4,24,26 Similarly, necrosis was not present in any of the kangaroos in this study, a finding that could reflect more chronic, low-grade hypertension. It was also possible that euthanasia precluded these changes or that, as appears to be the case in dogs and cats, fibrinoid necrosis is not a feature of the disease in M. fuliginosus.
Juxtaglomerular hyperplasia was also present in many kangaroos. In humans, juxtaglomerular hyperplasia was noted in conjunction with both renal arteriolar lesions and high blood pressure. 30 It has been proposed that decreased glomerular blood flow as a result of arteriolar hypertrophy and degeneration initiate juxtaglomerular cell proliferation and renin excretion in an attempt to increase blood pressure (and, subsequently, the glomerular filtration rate) via the renin-angiotensin system. 3 Interestingly, in the kangaroos severity of juxtaglomerular hyperplasia seemed to be independent of the arteriolar grade.
Blindness in several kangaroos was attributed to retinal detachment and atrophy. Retinal detachment is often associated with high indirect systolic blood pressures in domestic carnivores and humans and is referred to as hypertensive retinopathy. 12,19,20,25 The retina is very sensitive to changes in vascular dynamics by virtue of its high energy demand and the relatively limited space available for large blood vessels. Chorodial vascular damage that can occur by mechanisms similar to that described in renal arterioles. A loss of vascular integrity can lead to retinal ischemia, edema and hemorrhage into the subretinal space and, in severe cases, partial to complete retinal detachment. Histologic changes include fibrinoid necrosis and/or hypertrophy of the tunica media and, with chronicity, the development of pre-iridial fibrovascular membranes. Alternatively, or in addition, ischemia and subsequent detachment can result from autoregulatory vasoconstriction as an exaggerated response to hypertension. In the case of the latter, histologic vascular changes may not necessarily be present. 6,12,13,14 Although hyaline change was noted in retinal arterioles of one individual, for the most part, significant vascular-wall changes were lacking in the kangaroo eyes available for examination. Autoregulatory vasoconstriction may, therefore, be the mechanism of retinal detachment in the kangaroos. Acute onset and short duration before death may account for the lack of more chronic changes within the eye.
Several kangaroos had arterial and arteriolar hypertrophy within the central nervous system, and, in conjunction with the clinical neurologic signs, hypertensive encephalopathy was considered. The mechanism of hypertensive encephalopathy is not well understood. Several factors may play a role in the development of clinical signs, including arteriosclerosis and impaired cerebral blood vessel autoregulation, with subsequent cerebral edema. 4,13 A variety of neurologic signs, including seizures, ataxia, paresis, nystagmus, and disorientation, were observed in cats with poorly controlled hypertension. 4,19,20 Cerebral and cerebellar periarteriolar and interstitial edema were noted in one study that involved cats with reduced renal mass and systemic hypertension. In some of these cases, perivascular hemorrhage within the central nervous system was apparent on postmortem examination in association with hyperplastic arterioles, similar to that within the spinal cord of kangaroo No. 7. 4,19
Toxoplasmosis should be ruled out in any kangaroo with neurologic signs. Marsupials are particularly susceptible to infection with Toxoplasma gondii and often die without prior clinical signs. The most common histologic findings in macropods with active infection include nonsuppurative meningoencephalitis, myocarditis, and myositis with intralesional tachyzoites. 2,7 Although several animals had positive serologic titers, which suggested previous exposure, none had any histologic evidence of active toxoplasmosis. Toxoplasmosis, therefore, seems an unlikely cause of neurologic signs in any of the kangaroos.
Hypertension in domestic mammals is commonly associated with primary renal disease. Cause and effect can be difficult to determine, because it may be uncertain as to whether the renal changes are causing hypertension, are secondary to high blood pressures, or are a combination of the two. Among the kangaroos in this study, there was a distinct lack of renal lesions or blood work supportive of primary renal failure. As significant renal compromise must be present for secondary hypertension to develop, it is considered unlikely that primary renal disease was the cause of lesions among this group.
Some domestic felines with hypertension are diagnosed with hyperthyroidism. Although the exact mechanism has yet to be elucidated, it has been proposed that hyperthyroidism contributes to hypertension via β-adrenergic–induced increases in heart rate, contractility, systemic vasodilation, and subsequent activation of the renin-angiotensin-aldosterone system. A statistically significant correlation between hyperthyroidism and hypertension has not been established, and many cats with uncontrolled hyperthyroidism appear to have no associated ocular, renal, or neurologic changes. 19,20 In the two kangaroos with concurrent thyroid adenomas, renal arteriolar lesions were of the most severe grade. Circulating thyroid hormone levels were not assessed, and thus the impact of thyroid hormones, if any, is uncertain. Other animals had no histologic evidence of any thyroid abnormalities.
Because none of the common causes of secondary hypertension in domestic animals could be positively identified in these kangaroos, essential hypertension was considered. In humans, significant contributing factors have historically been nutrition (high salt intake), genetics, and stress; although, in many cases, the hypertension is ultimately considered idiopathic. 8 Hypertensive changes in woolly monkeys (Lagothrix lagotricha) have also been reported. In these animals, stress of captivity and/or age-related changes were among the speculated causes. 10 Stress levels among the kangaroos were taken into account as a possible contributor to hypertension. One relatively easily observed source is social stress. Kangaroo hierarchy dictates that one or few males are dominant, whereas other males and all females are considered to be low ranking. 23 Within this mob, during the reviewed time period, there were anywhere from 15 to 20 individuals, half of whom were males. Among animals with the most severe lesions, two were considered “low ranking” (based on keeper observations), and two were of uncertain status. Given that only 3 individuals over the 12-year time period were top-ranking males, no statistical significance could be established. In the past 5 years, many of the males have been separated from the mob. Although a relatively recent change, removal of the males has not resulted in an appreciable decline in hypertensive lesions. Captivity stress should also be considered. It is uncertain whether similar lesions occur in free-ranging western gray kangaroos.
A heritable predisposition seems unlikely, because only a few of the kangaroos with vascular changes were known to be related. Analysis of the diet as a cause of essential hypertension may merit further investigation to help determine whether salt intake could be an issue. Given that the average age of the kangaroos in the study (9.9 years) was high, with the average lifespan of female western gray kangaroos in captivity reported as 9–11 years, age as a contributor to vascular changes was considered. 11,18 The fact that there were at least minor arteriolar lesions or juxtaglomerular hyperplasia in all individuals could, to some degree, reflect normal changes associated with the age of the population or with the species; however, many of the common age-associated changes in humans are not present in these kangaroos. Although renal changes, such as arteriosclerosis and arterial wall hypertrophy, can increase significantly with age, small arterioles typically are spared. In addition, glomerulosclerosis, glomerular loss, tubular shortening, and marked interstitial fibrosis are common findings in aged human kidneys but were not appreciable among these kangaroos. 17,21
In summary, histologic changes within arterioles, the renal juxtaglomerular apparatus, and retinas of many kangaroos, in conjunction with clinical neurologic signs, were suggestive of hypertension. Common causes of secondary hypertension were not present, and essential (primary) hypertension was considered likely. Future research into potential causes or contributing factors may include assessment of thyroid hormone levels, more detailed analyses of parentage and diet, and antemortem blood pressure measurements.
Footnotes
Acknowledgements
We thank Jane Chladny and the University of Illinois Veterinary Diagnostic Laboratory's histology laboratory for slide preparation, Lou Ann Miller of the University of Illinois Center for Microscopic Imaging for assistance with electron microscopy, Dr. Kathleen Colegrove for reviewing the manuscript and the Zoological Pathology Program faculty, residents, and students who assisted with the necropsies included in this study. We would also like to thank Brookfield Zoo hospital staff for their essential support, and Mark Warneke and Glenn Granat for aid in compiling case materials.