Introduction

Chronic kidney disease (CKD) has been shown to be one of the most prevalent diseases in felid species, including domestic cats and many nondomestic felids.^1–3 Antemortem serum and urinary biochemical diagnosis of renal disease has been historically confirmed by high serum concentrations of urea nitrogen, creatinine, or phosphorus or high urine protein-to-creatinine ratio (UPC), alone or in combination, in concert with findings on urinalysis.⁴ Symmetric dimethylarginine (SDMA) is a product of protein metabolism resulting from the methylation of arginine protein residues, and > 90% of SDMA is excreted from the body by the kidneys,^5,6 making it a strong candidate for indirect assessment of kidney function and glomerular filtration rate (GFR).⁷ Although SDMA has not been specifically validated yet in tigers (Panthera tigris), it is showing great diagnostic value in several species, such as cats,⁸ dogs,⁹ and ferrets,¹⁰ for the earlier detection of renal disease. The sensitivity and specificity for the use of serum SDMA concentration measurements as a biomarker for a 30% decrease in GFR in domestic cats have been reported at 100% and 91%, respectively.⁸ When assessing kidney function, SDMA has several advantages over traditional renal biomarkers, such as creatinine and BUN. Although creatinine does not increase in dogs until there is a 75% decrease in GFR,¹¹ serum SDMA concentration has been shown to increase when there is as little as a 25% decrease and on average 40% decrease in kidney function in domestic cats,⁸ which allows for earlier detection of renal disease. In tigers, blood concentration of SDMA has been shown to increase approximately 3.6 months before blood concentration of creatinine,¹² giving SDMA a diagnostic preference for the early detection of renal disease in tigers. Additionally, creatinine is influenced by extrarenal factors such as endogenous production by muscle, meaning that a lack of lean muscle mass will falsely decrease creatinine concentration in patients with CKD.^13,14 As anorexia and cachexia are 2 common clinical findings in tigers with CKD,¹⁵ creatinine may not be the most sensitive or specific biomarker of renal disease in these animals. Additionally, BUN can be influenced by extrarenal factors that may affect the concentration, such as impaired hepatic function and poor nutritional status.¹⁶ These previous findings suggest that although the SDMA immunoassay has not yet been validated in tigers as it has in domestic cats¹⁷ and cheetahs,¹⁸ SDMA (compared with other traditional renal biomarkers) should be most closely connected to kidney function and declining GFR.

Diagnosis of CKD in domestic cats may also include imaging techniques (eg, ultrasonography) and palpation on physical examination.¹⁹ In tigers, however, ultrasonographic renal dimensions have been shown to be neither sensitive nor specific in the diagnosis of renal disease.²⁰ Complete physical examination of tigers, including kidney palpation, requires heavy sedation, complete anesthetization, or both which may or may not be feasible for some facilities and animals depending on any comorbidities and the associated risks of a full anesthetic event. This may mean that clinicians have to rely more heavily on nonspecific clinical signs of CKD in tigers, such as chronic, progressive weight loss, decreased appetite, or lethargy to create a clinical suspicion of disease. A diagnostic test that can give important information with as little intervention as possible would be ideal in investigating these nonspecific signs. Measurement of SDMA concentration has a practical advantage for surveillance of CKD given that undergoing phlebotomy may be trained in awake or lightly sedated tigers.

Common histologic renal changes in tigers have been described, with lymphocytic interstitial nephritis being the most prevalent renal lesion, reported in 36% (9/25) of tigers.²¹ These chronic, pathological changes to the kidneys presumptively result in a decrease in renal function; however, this has yet to be confirmed. As histologic evaluation of kidneys typically occurs after an animal has died, confirming a correlation between ≥ 1 renal biomarker and the severity of histologic changes may allow clinicians to better indirectly assess kidney function antemortem. With the previously mentioned idea that SDMA concentration in blood is most closely related to kidney function, we hypothesized that blood concentration of SDMA would more closely correlate with histologic kidney damage than would other renal biomarkers. This correlation and better understanding of kidney damage in the pathogenesis of CKD in tigers may further aid in the diagnosis, therapeutic management, and prognosis of these patients. The objective of the study reported here was to determine the utility of blood SDMA concentration measurement as a diagnostic tool for CKD in tigers by comparing results for SDMA with those for traditional renal biomarkers and investigating the correlations between these biomarkers and histopathologic kidney changes in tigers with CKD.

Materials and Methods

Postmortem examination and histopathology

Gross postmortem examinations were performed either at the UTVMC or the In-Sync facilities. For all tigers necropsied at the In-Sync facilities, sections of formalin-fixed tissues from all major organs were submitted to the UTVMC. Histologic evaluations were performed at the UTVMC. For each tiger that was euthanized or died, sections of kidney were routinely fixed in neutral-buffered 10% formalin for at least 48 hours, routinely processed, and cut as 5-μm-thin sections. Sections of kidney were stained with H&E, periodic acid–Schiff, and Masson’s trichrome stains for routine histopathologic evaluation.

A novel grading scale was created for the histologic assessment of these kidneys (Appendix) as an objective histologic kidney grading scale had not been validated in tigers. In domestic cats, it has been shown that tubular degeneration, interstitial inflammation, fibrosis, and glomerulosclerosis all increase as the International Renal Interest Society CKD stage increases.²³ In tigers, glomerular changes are uncommon, and tubulointerstitial nephritis, most commonly manifesting as interstitial inflammation, fibrosis, and tubular atrophy is by far the most dominant renal condition,²⁰ and as such, these features were selected for grading. Inflammation was graded at 20X magnification on a scale of 0 to 2, whereas fibrosis and tubular atrophy were each graded at 100X magnification on a scale of 0 to 3, for a total renal histologic score of 0 to 8. Inflammation was scored based on the percentage of inflammatory cells, predominantly lymphocytes and plasma cells, within the entire renal cortex stained with H&E and viewed at 2x power. A natural cutoff of 5% was used between inflammation scores of 1 and 2, as the vast majority of the cases exhibited inflammation affecting either less than or equal to 5% of the cortex (score = 1) or greater than 5% of the cortex (score = 2). Fibrosis was scored based on the percentage of fibrous tissue present within the renal cortex stained with Masson’s trichrome stain and viewed at 10x power. Tubular atrophy was scored based on the percentage of tubular atrophy present within the renal cortex stained with periodic acid–Schiff (PAS) stain and viewed at 10x power (Figure 1). Criteria for tubular atrophy grading included thickened basement membrane and irregular contour of renal tubular margins. Ten fields (100X magnification) that were representative of the renal cortex as a whole were chosen to assess for fibrosis and tubular atrophy. The percentage cutoffs for each score of fibrosis and tubular atrophy corresponded most consistently with mild (score = 1), moderate (score = 2), or severe (score = 3) degree of fibrosis or tubular atrophy as assessed by a board-certified anatomic pathologist (MMS). If a specimen had > 1 kidney sample and those sections had different scores, a mean score was calculated and used.

Representative photomicrographs of histologic sections of kidneys from 35 adult tigers (Panthera tigris) that died or were euthanized at either Tiger Haven in Kingston, Tennessee or In-Sync Exotics Wildlife Rescue & Educational Center in Wylie, Texas between June 2016 and June 2020, organized by category and score of renal damage evaluated: inflammation (A, B, and C), fibrosis (D, E, F, and G), and tubular atrophy (H, I, J, and K). A— Histologically normal renal cortex with an inflammation score of 0 (no inflammatory cells visible). H&E stain; bar = 500 µm. B—Small clusters of lymphocytes (arrowhead) expand less than 5% of the renal cortical interstitium: inflammation score of 1. H&E stain; bar = 500 µm. C—Larger clusters of inflammatory cells (arrowhead) expand more than 5% of the renal cortical interstitium: inflammation score of 2. H&E stain; bar = 500 µm. D—Histologically normal renal cortex with normal fibrous connective tissue highlighted blue with Masson trichrome stain: fibrosis score of 0 (fibrosis affects 0% to 9% of renal cortex). Masson trichrome stain; bar = 100 µm. E—Fibrosis composes approximately 10% of the renal cortex field: fibrosis score of 1 (fibrosis affects 10% to 19% of renal cortex). Masson trichrome stain; bar = 100 µm. F—Fibrous connective tissue composes approximately 25% of the renal cortex field: fibrosis score of 2 (fibrosis affects 20% to 29% of renal cortex). Masson trichrome stain; bar = 100 µm. G—Fibrous connective tissue effaces and replaces tubules, composing nearly 80% of the field: fibrosis score of 3 (fibrosis affects 30% or greater of renal cortex). Masson trichrome stain; bar = 100 µm. H—Histologically minimally affected renal cortex: tubular atrophy score of 0 (tubular atrophy affects 0% to 9% of renal cortex). Rare tubules have decreased luminal diameter and thickened basement membranes, highlighted magenta (Note: basement membranes of tubules, the glomeruli, and glomerular capillaries stain magenta with PAS stain). PAS stain; bar = 100 µm. I—Approximately 15% of tubules have decreased tubular diameter: tubular atrophy score of 1 (tubular atrophy affects 10% to 19% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. J—Approximately 25% of the tubules within the field have decreased to absent lumens and thickened basement membranes: tubular atrophy score of 2 (tubular atrophy affects 20% to 29% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. K—Greater than 50% of renal tubules are absent as evidenced by a complete lack of basement membrane staining (arrowhead): tubular atrophy score of 3 (tubular atrophy affects 30% or greater of renal cortex). PAS stain; bar = 100 µm.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Representative photomicrographs of histologic sections of kidneys from 35 adult tigers (Panthera tigris) that died or were euthanized at either Tiger Haven in Kingston, Tennessee or In-Sync Exotics Wildlife Rescue & Educational Center in Wylie, Texas between June 2016 and June 2020, organized by category and score of renal damage evaluated: inflammation (A, B, and C), fibrosis (D, E, F, and G), and tubular atrophy (H, I, J, and K). A— Histologically normal renal cortex with an inflammation score of 0 (no inflammatory cells visible). H&E stain; bar = 500 µm. B—Small clusters of lymphocytes (arrowhead) expand less than 5% of the renal cortical interstitium: inflammation score of 1. H&E stain; bar = 500 µm. C—Larger clusters of inflammatory cells (arrowhead) expand more than 5% of the renal cortical interstitium: inflammation score of 2. H&E stain; bar = 500 µm. D—Histologically normal renal cortex with normal fibrous connective tissue highlighted blue with Masson trichrome stain: fibrosis score of 0 (fibrosis affects 0% to 9% of renal cortex). Masson trichrome stain; bar = 100 µm. E—Fibrosis composes approximately 10% of the renal cortex field: fibrosis score of 1 (fibrosis affects 10% to 19% of renal cortex). Masson trichrome stain; bar = 100 µm. F—Fibrous connective tissue composes approximately 25% of the renal cortex field: fibrosis score of 2 (fibrosis affects 20% to 29% of renal cortex). Masson trichrome stain; bar = 100 µm. G—Fibrous connective tissue effaces and replaces tubules, composing nearly 80% of the field: fibrosis score of 3 (fibrosis affects 30% or greater of renal cortex). Masson trichrome stain; bar = 100 µm. H—Histologically minimally affected renal cortex: tubular atrophy score of 0 (tubular atrophy affects 0% to 9% of renal cortex). Rare tubules have decreased luminal diameter and thickened basement membranes, highlighted magenta (Note: basement membranes of tubules, the glomeruli, and glomerular capillaries stain magenta with PAS stain). PAS stain; bar = 100 µm. I—Approximately 15% of tubules have decreased tubular diameter: tubular atrophy score of 1 (tubular atrophy affects 10% to 19% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. J—Approximately 25% of the tubules within the field have decreased to absent lumens and thickened basement membranes: tubular atrophy score of 2 (tubular atrophy affects 20% to 29% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. K—Greater than 50% of renal tubules are absent as evidenced by a complete lack of basement membrane staining (arrowhead): tubular atrophy score of 3 (tubular atrophy affects 30% or greater of renal cortex). PAS stain; bar = 100 µm.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Representative photomicrographs of histologic sections of kidneys from 35 adult tigers (Panthera tigris) that died or were euthanized at either Tiger Haven in Kingston, Tennessee or In-Sync Exotics Wildlife Rescue & Educational Center in Wylie, Texas between June 2016 and June 2020, organized by category and score of renal damage evaluated: inflammation (A, B, and C), fibrosis (D, E, F, and G), and tubular atrophy (H, I, J, and K). A— Histologically normal renal cortex with an inflammation score of 0 (no inflammatory cells visible). H&E stain; bar = 500 µm. B—Small clusters of lymphocytes (arrowhead) expand less than 5% of the renal cortical interstitium: inflammation score of 1. H&E stain; bar = 500 µm. C—Larger clusters of inflammatory cells (arrowhead) expand more than 5% of the renal cortical interstitium: inflammation score of 2. H&E stain; bar = 500 µm. D—Histologically normal renal cortex with normal fibrous connective tissue highlighted blue with Masson trichrome stain: fibrosis score of 0 (fibrosis affects 0% to 9% of renal cortex). Masson trichrome stain; bar = 100 µm. E—Fibrosis composes approximately 10% of the renal cortex field: fibrosis score of 1 (fibrosis affects 10% to 19% of renal cortex). Masson trichrome stain; bar = 100 µm. F—Fibrous connective tissue composes approximately 25% of the renal cortex field: fibrosis score of 2 (fibrosis affects 20% to 29% of renal cortex). Masson trichrome stain; bar = 100 µm. G—Fibrous connective tissue effaces and replaces tubules, composing nearly 80% of the field: fibrosis score of 3 (fibrosis affects 30% or greater of renal cortex). Masson trichrome stain; bar = 100 µm. H—Histologically minimally affected renal cortex: tubular atrophy score of 0 (tubular atrophy affects 0% to 9% of renal cortex). Rare tubules have decreased luminal diameter and thickened basement membranes, highlighted magenta (Note: basement membranes of tubules, the glomeruli, and glomerular capillaries stain magenta with PAS stain). PAS stain; bar = 100 µm. I—Approximately 15% of tubules have decreased tubular diameter: tubular atrophy score of 1 (tubular atrophy affects 10% to 19% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. J—Approximately 25% of the tubules within the field have decreased to absent lumens and thickened basement membranes: tubular atrophy score of 2 (tubular atrophy affects 20% to 29% of renal cortex). Note the particularly affected region (arrowhead). PAS stain; bar = 100 µm. K—Greater than 50% of renal tubules are absent as evidenced by a complete lack of basement membrane staining (arrowhead): tubular atrophy score of 3 (tubular atrophy affects 30% or greater of renal cortex). PAS stain; bar = 100 µm.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Results

Blood samples were obtained from 86 tigers, of which, 36 either died naturally or were euthanized due to quality-of-life considerations before the end of June 2020. Of these 36 tigers, 33 had necropsy performed by the UTVMC anatomic pathology necropsy service. One of these tigers had severe postmortem autolysis due to an extended interval which eliminated the ability to assess gross and histologic pathological changes to the kidneys. The 3 remaining tigers were necropsied at the In-Sync facilities, and sections of formalin-fixed tissues from all major organs were submitted to the UTVMC. These 35 tigers ranged in age from 7 to 23 years (mean ± SD, 16.6 ± 3.81 years).

Thirty blood samples were analyzed without having been frozen first, and 5 samples had been frozen and stored in a –70 °C freezer from immediately after obtaining the serum or heparinized plasma sample (after centrifugation) to the time of sample analysis. Samples had been frozen between 1 week and 10 months before shipment to the testing laboratory, and upon arrival to the laboratory, the samples remained frozen in a –70 °C freezer for up to 18 months before further analysis. Urine was sampled from 21 of these 35 tigers for urinalysis alone (n = 7), UPC alone (n = 9), or both (n = 5).

Ten of the 36 tigers that died or were euthanized had > 1 kidney sample available, and 6 of those 10 had differing histologic kidney grading scores. The mean was calculated from these samples and reported as the score for that animal. The number of days between the sampling of blood and urine and the death or euthanasia of tigers ranged between 0 and 662 days (mean ± SD, 53.0 ± 130.13 days). Results for kidney score versus renal biomarkers were plotted (Figure 2; Supplementary Figures S1–S3). Results of Spearman rank correlation coefficient analyses indicated that SDMA concentration was the renal biomarker with the greatest correlation (ρ = 0.667, P < 0.001) with histologic kidney score. Blood creatinine concentration (ρ = 0.624, P < 0.001), BUN concentration (ρ = 0.588, P < 0.001), and USG (ρ = –0.639, P = 0.025) all had significant correlations with kidney score, whereas the blood phosphorus concentration (ρ = 0.220, P = 0.205), urine protein concentration (ρ = –0.084, P = 0.795), and the UPC (ρ = 0.320, P = 0.265) did not. The positive correlations of SDMA, BUN, and creatinine concentrations with kidney score, indicated that these values increased as kidney score increased and vice versa. The negative correlation for USG meant that this value increased as kidney score decreased and vice versa. Results of Spearman rank correlation coefficient analyses for SDMA concentration and kidney damage score indicated that SDMA significantly (P < 0.001) correlated with all 3 categories histologic changes: inflammation (ρ = 0.559), fibrosis (ρ = 0.692), and tubular atrophy (ρ = 0.616).

Scatterplots of the associations between histologic kidney score and blood (serum or plasma) concentration of symmetric dimethylarginine (SDMA; A), creatinine (B), or BUN (C) or urine specific gravity (D; n = 12) for the 35 tigers described in Figure 1. For each plot, each circle represents the results for 1 tiger, and the dotted line represents the line of best fit.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Scatterplots of the associations between histologic kidney score and blood (serum or plasma) concentration of symmetric dimethylarginine (SDMA; A), creatinine (B), or BUN (C) or urine specific gravity (D; n = 12) for the 35 tigers described in Figure 1. For each plot, each circle represents the results for 1 tiger, and the dotted line represents the line of best fit.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Scatterplots of the associations between histologic kidney score and blood (serum or plasma) concentration of symmetric dimethylarginine (SDMA; A), creatinine (B), or BUN (C) or urine specific gravity (D; n = 12) for the 35 tigers described in Figure 1. For each plot, each circle represents the results for 1 tiger, and the dotted line represents the line of best fit.

Citation: Journal of the American Veterinary Medical Association 260, 13; 10.2460/javma.21.04.0216

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Discussion

The objective of this study was to determine the utility of blood SDMA concentration measurement as a diagnostic tool for CKD in tigers by comparing results for SDMA with those for traditional renal biomarkers and investigating the correlations between these biomarkers and histopathologic kidney changes in tigers with CKD. Through this study, it was determined that SDMA most strongly, and positively correlated with kidney score, using the proposed scoring system, among the renal biomarkers measured. As the kidney score assessed histologic kidney damage, these findings supported our hypothesis that blood concentration of SDMA would more closely correlate with histologic kidney damage than would other renal biomarkers. Creating an objective method for pathologists to utilize in tigers was desirable to identify the most accurate relationships between histologic evaluation of kidneys and measured renal biomarkers. These findings may allow clinicians to better assess kidney damage in patients antemortem and benefit pathologists and clinicians by allowing for more uniform and repeatable histologic assessments of kidneys from tigers with CKD.

When comparing the relationships between kidney score and renal biomarkers in the present study, SDMA had the highest correlation with histologic changes to the kidneys in tigers (ρ = 0.667, P < 0.001). This relationship provides further clarity for antemortem assessment of patients with renal disease. Because the physiologic actions of the kidney rely on structural function, chronic pathological changes to the kidney should presumptively lead to an overall clinical decline in that patient. Confirming SDMA correlates with severity of histologic disease provides additional evidence when staging kidney disease and assist in providing a long-term prognosis in tigers with suspect or diagnosed CKD. As expected, SDMA also significantly correlated with each individual criteria of total kidney score. Because CKD is typically a multifactorial disease with a tight interplay between interstitial and tubular compartments rather than a disease of 1 particular pathological change, all 3 categories of histopathologic renal changes (ie, inflammation, fibrosis, and tubular atrophy) were expected to play a role in the pathogenesis of CKD in tigers. This would suggest that increases in SDMA would correlate with increasing severity of each change, which was supported by our findings.

Confirming SDMA as a strong biomarker for renal disease is particularly appealing as tigers can be trained to tolerate phlebotomy under only light sedation, which allows for the possibility to avoid full anesthetic events. This is especially important for tigers with comorbidities that contraindicate or complicate general anesthesia, while also keeping clinicians and caregivers safe. If a tiger can be trained to present itself at a barrier for phlebotomy, the need for clinicians and caregivers to be in the same enclosure as the tiger for assessment of suspected CKD would no longer be necessary. Even if diagnostic modalities, such as renal palpation and ultrasonography, were proven to be more sensitive, specific, or both in the diagnosis of CKD in tigers, those methods require much more time spent in close proximity to the animal, which comes with safety concerns even in anesthetized or heavily sedated tigers.

Traditional renal biomarkers for CKD, such as BUN and creatinine, also had significant (P < 0.001) correlation (ρ = 0.588 and ρ = 0.624, respectively) with kidney score as expected with the method used in the present study. Of the measured urine biomarkers, USG was the only one that correlated significantly (ρ = –0.639, P = 0.025) with kidney score. The negative correlation coefficient between kidney score and USG is supported clinically by the notion that as the kidney becomes more damaged and starts to lose functioning nephrons, the ability to concentrate urine will decrease, leading to a decreased USG.²⁴ The lack of a significant correlation between kidney score and other renal biomarkers, such as UPC or urine concentrations of phosphorus or protein, in the present study may incline clinicians to potentially reprioritize results when assessing tigers for CKD, focusing instead on the biomarkers that our findings indicated significantly correlated with kidney score.

For anatomic pathologists examining samples from captive tigers, this proposed grading scale provides an objective method to assess histologic damage to the kidneys in tigers with CKD. This scale accounts for the major and most frequently encountered postmortem lesions of CKD described in tigers to facilitate a repeatable and clinically relevant assessment process across pathologists, minimizing differences in sampling. The objectivity of this scale may minimize the bias between evaluators and may lead to more consistent reporting of the severity of postmortem findings.

One confounding finding was the combination of a blood SDMA concentration of 100 μg/dL and a kidney score of 1 for 1 tiger. When considering the explanation of this outlier, sampling bias during gross examination should be considered. In a grossly abnormal kidney, a section of kidney that has the gross lesion or lesions will naturally be chosen for histologic evaluation along with a section that looks more grossly normal (MM Sula, DVM, College of Veterinary Medicine, University of Tennessee, email, July 23, 2020). In a kidney that is absent of any obvious gross lesions, a sample for histologic evaluation must be taken at the discretion of the prosector, which may or may not be a representative sample of the kidney as a whole. In domestic cats, some gross abnormalities of the kidneys more specific for CKD are typical, such as a decrease in overall size and pitting of the surface of affected kidneys.²⁵ In dogs, common gross postmortem findings of CKD include altered size and shape and difficulty removing the capsule.²⁶ The area where gross changes to the kidney are present would then be a site of sampling for histologic evaluation. Specific gross lesions of CKD in tigers are not as well documented as they are in domestic cats. In a previous study,²⁰ it was shown that ultrasonographic renal measurements were neither sensitive nor specific in the diagnosis of CKD in tigers. This suggests that, unlike a domestic cat, a tiger with CKD and a tiger without CKD may have kidneys of the same size and shape. This holds true to the authors’ experience, where it is common, in tigers, for gross changes to be absent in a kidney that is moderately to severely affected by CKD (Supplementary Figure S4), this potentially leads to sampling bias. In these grossly normal kidneys, a sample taken at random may not be histologically representative, which will lead to a falsely low kidney score in a patient with increased SDMA or other renal biomarkers due to CKD. This was considered the likely scenario in the outlier kidney of this study.

Additionally, these data lead to opportunities for further research into how early SDMA increases in the pathogenesis of CKD relative to other renal biomarkers, which has been documented in other felid species such as domestic cats and cheetahs.^17,18 This, along with the findings of this study, will further clarify the biological aspect of renal disease in tigers and allow for clinicians to better assess and treat tigers for CKD.

Limitations of the present study included the lack of a consistent interval between the dates of sample collection and necropsy. This would be desirable for future studies when attempting to correlate the severity of clinical disease and renal histologic changes. A greater number of urine samples would also affect the power of the correlation coefficients for USG, urine protein, and UPC as well as further evaluation of confounding factors like lower urinary infection or inflammation leading to post-renal proteinuria. Greater understanding of the correlation between severity of clinical signs and pathological changes may grant clinicians greater insight into each patient’s condition based on clinical presentation and progression. Extensive antemortem clinical data, such as clinical signs and their degree of severity, or specific treatments or medications prescribed for the treatment of CKD in an individual, were not readily available for each animal in this study. In part, this was due to the generally nonspecific clinical signs, such as weight loss and inappetence, most commonly attributed to CKD. Restricted clinical evaluation and therapeutic intervention, due to limited ability to closely handle and examine these animals, further complicates detailed clinical staging of these animals. Although any relevant clinical findings should be examined in each individual case, SDMA may provide clinicians with the best antemortem assessment of kidney function in tigers. Postmortem examination of kidneys may also illuminate the burden of clinical disease after an animal has died or been euthanized, so that clinicians may refine the interpretation of physical examination and more subjective clinical findings. Additionally, the relationship between the severity of clinical CKD and more detailed histologic kidney evaluation in tigers requires further investigation. The use of more extensive microscopic examination including immunofluorescence and transmission electron microscopy (TEM) would certainly have been desirable in this study for complete histologic evaluation of the kidneys. However, the pathogenesis of CKD in domestic cats more commonly involves glomerular changes, such as glomerulosclerosis, than in tigers, which gives immunofluorescence and TEM greater diagnostic value in domestic cats for the diagnosis of renal disease.^27,28 Additionally, these modalities are largely considered too expensive for routine diagnostic evaluation in nondomestic species. For these reasons, these more advanced techniques were not performed as part of the present study. Further investigation is warranted to discover how immunofluorescence and TEM may assist in the diagnosis of CKD in tigers. A final limitation, as previously mentioned, was that the SDMA immunoassay was not yet validated in tigers as it is in other felid species. We, however, expected a similar increase of SDMA in the pathogenesis of CKD in tigers as that of other felid species. These additional studies and deeper investigation may increase the utility of SDMA in the diagnosis and management of CKD in tigers. As the relationship between clinical disease, clinical biomarkers, and histologic findings are more thoroughly realized, we will better develop our understanding of how this condition evolves in these animals.

For clinicians that work with large exotic animals, diagnostic procedures can provide an extra layer of difficulty, compared with those in domestic animals. This results in the need to justify every diagnostic procedure as a worthwhile investment. In suspected cases of CKD or for simple monitoring of disease progression in tigers, SDMA was shown in the present study to have been the most effective among renal biomarkers evaluated for antemortem, indirect assessment of histologic kidney damage while potentially being measurable with substantially less risk to the animals and caretaking personnel. This finding was supported by a grading scale that can be replicated across many patients and pathologists for more consistent postmortem assessment of CKD in tigers. Standardizing this evaluation will allow for more effective communication from pathologists to clinicians about the severity of damage and may be useful as further studies investigate how these changes relate to clinical disease.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org