Assessment of serum symmetric dimethylarginine and creatinine concentrations in hyperthyroid cats before and after a fixed dose of orally administered radioiodine

Abstract Background Serum symmetric dimethylarginine (SDMA) is a sensitive renal biomarker for detecting early chronic kidney disease (CKD) in nonhyperthyroid cats, but knowledge regarding its performance in hyperthyroid cats remains limited. Objectives To determine the relationship between serum SDMA, creatinine and total thyroxine (TT4) concentrations in hyperthyroid cats before (T0) and 3 months after (T1) receiving a PO fixed dose of radioiodine. Animals Eighty client‐owned hyperthyroid cats. Methods Prospective cohort study. Serum TT4, and SDMA, creatinine concentrations, and urine specific gravity were measured at T0 and T1. Nonparametric tests were used to determine the relationship among SDMA, and creatinine and TT4 concentrations. Agreement between SDMA and creatinine regarding CKD staging at both time points was assessed using Goodman and Kruskal's gamma statistic. Results Mean serum SDMA concentration increased after treatment of hyperthyroidism. However, 21 of 75 cats experienced a decrease in SDMA between T0 and T1, whereas creatinine decreased in only 2 cats. A moderate correlation between SDMA and creatinine was seen at T1 (r = 0.53; P < .001) but not at T0 (r = 0.13; P = .25). Where assessable at T1, poor agreement was observed between SDMA and creatinine and CKD stage (Goodman and Kruskal's gamma 0.20; P = .29). Conclusions and clinical importance Discordant outcomes between SDMA and creatinine after radioiodine treatment in cats with hyperthyroidism suggest extrarenal factors may interfere with the reliability of SDMA to adequately reflect renal function. As a result, SDMA should not be interpreted in isolation in hyperthyroid cats treated with radioiodine.


| INTRODUCTION
Hyperthyroidism and chronic kidney disease (CKD) are common in older cats. Previous studies indicate that 10% to 49% of cats with hyperthyroidism have concurrent renal azotemia diagnosed before or after treatment. [1][2][3][4][5][6] Chronic kidney disease has implications for the clinical decision-making process regarding optimal treatment options for hyperthyroidism and may decrease the lifespan of affected cats.  7 The increase in thyroid hormone production in hyperthyroidism increases renal blood flow and glomerular capillary hydrostatic pressure, thereby increasing glomerular filtration rate (GFR). Indirect measures of GFR such as serum creatinine and urea nitrogen concentrations are notoriously inaccurate at detecting CKD in hyperthyroid cats. Their inaccuracy is largely attributable to the effects of extrarenal factors such as muscle mass, as well as daily variability in exogenous and endogenous protein loads. [8][9][10][11] Pretreatment GFR has been proposed as a potential predictor of post-treatment azotemia. 5,12,13 However, sensitive and specific cutoffs have proved difficult to establish. 5,12 Although single sample plasma clearance methods have been validated in cats, these are yet to be assessed in hyperthyroid cats and currently are not widely available in the clinical setting. 14,15 Symmetric dimethylarginine (SDMA) is an indirect biomarker of GFR that is unaffected by muscle mass and reportedly detects CKD with higher sensitivity than creatinine. 11,16 Recently, SDMA has been incorporated as an adjunctive variable in the International Renal Interest Society (IRIS) guidelines for CKD staging and management. 17 However, the influence of hyperthyroidism on SDMA in cats is yet to be explored in detail.
It has been reported that increases in serum SDMA concentration in nonazotemic hyperthyroid cats before treatment are relatively insensitive, but specific for predicting azotemia after treatment. 18 19 Further exploration of the relationships among SDMA, creatinine and TT4 in another population of hyperthyroid cats is warranted.
Our objectives were to prospectively measure serum SDMA concentrations in hyperthyroid cats receiving a fixed PO dose of radioiodine before and after treatment and to assess serum SDMA concentration in relation to serum creatinine and TT4 concentrations at both time points.
It was hypothesized that serum SDMA concentration would increase significantly as hyperthyroidism resolves. Serum SDMA and creatinine concentrations were expected to be significantly correlated only after hyperthyroidism resolved, leaving fewer extrarenal factors to affect serum creatinine concentration. After radioiodine treatment, where renal dysfunction was suspected based on serum creatinine concentration and concurrent suboptimal urine specific gravity (USG), serum SDMA concentration was hypothesized to be concurrently increased.

| Sample population
This study was a prospective cohort study of client-owned cats with a confirmed diagnosis of hyperthyroidism and deemed appropriate for treatment with a fixed (approximately 138Mbq; 3.7 mCi) PO dose of radioiodine at the University of Melbourne's U-Vet Werribee Animal Hospital. The enrollment period was from January 2017 to September 2018. All procedures included in the study were approved by the Animal Ethics Committee of the University of Melbourne (AEC application ID 1613858) and all owners gave written consent to have their cats included in the study.
To be eligible for inclusion in the study, cats were required to meet the inclusion and exclusion criteria of a previously established standard radioiodine treatment protocol. 20 Cats were eligible for treatment with fixed dose radioiodine after a definitive diagnosis of hyperthyroidism was made based on presence of clinical signs consistent with hyperthyroidism (eg, weight loss despite polyphagia, polydipsia and polyuria, unkempt coat) 1 and serum TT4 concentration above the upper limit of the reference interval.

| Data and sample collection
Age, sex and neuter status were recorded for each cat. At initial presentation (T0) and at least 3 months (T1) after radioiodine treatment, historical information collected included any clinical signs attributable to hyperthyroidism. 21 All cats were examined by the primary clinician; either a resident or board-certified (ACVIM or ECVIM-CA) specialist in small animal internal medicine. The physical examination included blood pressure measurement using Doppler sphygmomanometry, 22 body weight, body condition score (BCS), and muscle condition score (MSC). The BCS was assigned by a validated 9-point system, 23 whereas the MCS was assigned by a 4-point system. 24 Blood and urine (voided or cystocentesis collection) were sampled at T0 and T1 to measure clinicopathological variables as outlined below. Where possible, samples were collected from unsedated cats, but fractious cats had sample collection performed under IM sedation.
Samples that were collected after sedation were flagged, and the type and amount of sedation used were recorded.

| Clinicopathological assays
Blood collected into serum tubes was centrifuged within 1 hour of collection and serum separated for standard biochemistry analysis within 2 hours of collection at the clinical pathology laboratory of the U-Vet Animal Hospital by standardized methods. Laboratory technicians manually read the packed cell volume (PCV) after centrifugation of blood in hematocrit tubes, and determined the total protein concentration of the serum and USG with a handheld, calibrated refractometer.
The creatinine assay was a standard kinetic colorimetric assay based on the Jaffé method, (CREJ2, Roche Diagnostics Ltd, Switzerland) and the urea assay was a standard kinetic colorimetric assay based on urease and glutamate dehydrogenase. (UREAL, Roche Diagnostics Ltd, Switzerland).
At T0, the remaining serum was separated in 0.25 mL aliquots and stored at −80 C until the T1 sample was acquired. At this time, the T0 samples were thawed to allow paired analysis of T0 and T1 samples for TT4 and SDMA concentrations. Serum TT4 concentration was measured using a solid-phase competitive chemiluminescence immunoassay (Immulite 1000 XPI, Siemens, Victoria, Australia), previously validated for use in cats. 25 The reference interval for the TT4 assay was 7.

| Defining kidney dysfunction
Renal azotemia was defined as a serum creatinine concentration of ≥1.6 mg/dL (≥140 μmol/L) or a serum SDMA concentration of ≥18 μg/dL with concurrent USG <1.035. 27,28 The severity of kidney dysfunction then was stratified separately into CKD stages based on serum creatinine and SDMA concentrations according to IRIS CKD staging guidelines. 27 Where urine concentration was ≥1.035, prerenal azotemia was defined as a concurrent serum creatinine concentration ≥1.6 mg/dL (≥140 μmol/L) or serum SDMA concentration ≥14 μg/dL.

| Sample characteristics
Based on sample size calculations, data from at least 76 cats were required for the study to have sufficient power to detect a difference between SDMA and creatinine for detection of CKD at T1. A dropout rate up to 40% was expected, and thus in the period of inclusion, 108 cats treated with fixed dose radioiodine were enrolled with owner consent. The mean calibrated dose of PO radioiodine used was 3.6 mCi (131.6 MBq; SD, 0.1 mCi or 5.0 MBq). Twenty-eight (26%) cats did not complete the study or were excluded: owners of 16 cats declined follow-up examinations at the study institution because of travel distance, owners of 11 cats could not be contacted and 1 cat was diagnosed with large cell gastrointestinal lymphoma before T1.
Eighty (74%) cats returned for their T1 evaluation and were included in the study. The median duration from treatment to re-evaluation was 108 days (IQR, 99-134 days).
Mean body weight, BCS and MCS increased, and mean systolic blood pressure decreased significantly after treatment (Supporting Information Table S1). At T0, 17 cats had serum TT4 concentrations >15.0 μg/dL (>193.0 nmol/L). At T1 (after radioiodine treatment), 2 (2.5%) cats had persistent hyperthyroidism based on serum TT4 concentrations above the upper limit of the reference interval. Sixty-six (82.5%) cats had serum TT4 concentrations within the reference interval and 12 (15%) cats had serum TT4 concentrations below the lower limit of the reference interval.

| Renal clinicopathological parameters before and after radioiodine treatment
In the total sample population of 80 cats, PCV decreased and total protein concentration increased significantly at T1 compared to T0. A significant increase in the means of serum creatinine, urea and SDMA occurred, whereas mean serum TT4 and USG decreased significantly in the total sample at T1 (Table 1). Similarly, when only cats with serum TT4 concentrations within the reference interval (ie, those considered euthyroid) at T1 were considered (n = 66 cats), a significant increase in mean serum creatinine, urea and SDMA concentrations occurred from T0 to T1, whereas the mean USG decreased significantly from T0 to T1 (Supporting Information Table S2). In this group of cats, 32 of 62 (51%) had USG < 1.035 at T1, of which 21 cats (34% of the subgroup) had renal azotemia based on the serum creatinine concentration and USG.
Ten of these 21 azotemic cats had been nonazotemic with USG < 1.035 at T0. Five cats had USG < 1.035 at both time points but remained nonazotemic. Only 2 cats with USG <1.035 at T0 had an increase in USG to ≥1.035 at T1, neither of these cats had renal azotemia based on the serum creatinine concentration at T0.
In the 12 cats with a serum TT4 concentration below the lower limit of the reference interval at T1, mean serum SDMA concentrations were not significantly different between T0 (16.0 μg/dL; SD, 13.10) and T1 (12.3 μg/dL; SD, 3.9; P = .36. [paired t test]). However, mean serum concentrations for creatinine and urea increased whereas mean USG decreased significantly from T0 to T1 (Supporting Information - Table S3). Five (41.6%) of 12 cats were considered to have renal azotemia based on the serum creatinine concentration and USG at T1, in which USG < 1.035 had been present at T0 in 2 of 5 cats.
In the 2 cats that remained hyperthyroid at T1, both cats had USG ≥1.035, with normal serum creatinine concentrations at T0 and T1. The serum SDMA concentrations were within the normal reference interval for both cats at T0. At T1, the serum SDMA concentration remained normal 1 cat, and increased slightly above the reference interval in the other.

| Categorization of renal dysfunction by SDMA or creatinine before and after radioiodine treatment
The categorization of IRIS stages at T0 based on both serum creatinine and SDMA concentration, is summarized in Table 2 The categorization of IRIS stage based on serum creatinine or SDMA concentration, respectively, at T1 is summarized in Table 3.
Seventy-four (93%) of 80 cats had complete renal clinicopathological information including serum SDMA and creatinine concentrations and USG. As mentioned previously, 3 cats had no serum SDMA concentration measured and 4 cats had no USG measured; these were recorded as missing values in Table 3 Table 3). None of the cats with discordant staging results received sedation at T1.
For all cats at T1, poor agreement of IRIS CKD staging was found when comparing classification by creatinine with SDMA; the Goodman and Kruskal's gamma was 0.20 and did not achieve statistical significance (P = .29).

T A B L E 1
Clinicopathologic data of 80 hyperthyroid cats before (T0) and after (T1) radioiodine treatment Note: International Renal Interest Society CKD staging (0-4) according to the creatinine (columns) and SDMA (rows) concentrations. The allocation of stage 0 is given to cats with adequate urine concentration (urine specific gravity ≥1.035) and deemed to have adequate renal function. The allocation of possible stage 1 is given to cats with urine specific gravity <1.035, but with creatinine <140 μmol/L and/or SDMA <18 μg/dL. Bolded numbers indicate agreement between serum creatinine and SDMA. Abbreviations: CKD, chronic kidney disease; SDMA, symmetric dimethylarginine.  Tables S4 and S5).

| Relationships among serum SDMA, creatinine, and TT4 concentrations
The relationship between SDMA and TT4 and between creatinine and TT4 at T0 and T1 was assessed in 77 and 80 cats, respectively.
No significant correlation was found between serum SDMA and

| DISCUSSION
We prospectively assessed serum SDMA, creatinine and TT4 concentrations in a sample population of 80 Australian cats before and after F I G U R E 2 Correlation between serum creatinine and serum symmetric dimethylarginine (SDMA) in 76 cats after radioiodine treatment, excluding the outlier Note: Each dot represents 1 cat. The solid line represents the line of best fit. treatment of hyperthyroidism with a PO fixed dose of radioiodine. Consistent with the study hypothesis and recently published results, 18 mean SDMA concentration increased significantly as hyperthyroidism resolved.
This is presumably because serum SDMA concentration correlates with GFR 16,29 and an increase in serum SDMA concentration is expected as the effects of hyperthyroidism on renal perfusion resolve. 5,30,31 However, serum SDMA concentration did not increase in 21 (28%) of 75 cats, 5 of which had serum TT4 concentrations below the reference interval at T1. In these cats, the concurrent decrease in serum SDMA concentration is particularly surprising because several of these cats may have been hypothyroid, and GFR is expected to decrease in approximately half of cats with iatrogenic hypothyroidism. 32,33 Similar to previous studies, SDMA and creatinine were not correlated before radioiodine treatment, with only moderate correlation seen after treatment. 18 [38][39][40] Apart from the abovementioned extrarenal factors, another study proposed that serum SDMA concentrations in hyperthyroid cats may be affected by alterations in protein metabolism and potential alterations in hepatic clearance of SDMA. 19 Upregulation of SDMA production also is a possible reason, but so far, it has been reported only in growing animals. 41  Our study sample population is comparable to that of recent studies assessing renal parameters before and after radioiodine treatment in cats 18,19,44 ; namely, geriatric, predominantly domestic breed, hyperthyroid cats. Female neutered cats were slightly overrepresented in our study, but it also has been observed in other studies. 18,19 The proportions of cats with apparent decreased renal function at T0 and T1 are similar to existing reports in the literature, 1,2,3,7,45 and as previously reported, renal azotemia was more common in cats with serum TT4 concentrations below the lower limit of the reference interval after radioiodine treatment than in those with serum TT4 concentrations within the reference interval. 18,44 The chosen follow-up time after radioiodine treatment was approximately 3 months. Although the expected recovery time for atrophic thyroid tissue after resolution of hyperthyroidism is 1 to 3 months in most cats, 46-48 recent evidence indicates a large proportion of cats could develop overt hypothyroidism as late as 6 to 12 months after radioiodine treatment. 32 This could further influence renal function such that results of our study may not accurately reflect long-term thyroid function in cats. However, most changes in renal function related to resolution of hyperthyroidism occur in the first month after radioiodine treatment, with no further significant change in GFR between 1 and 6 months after treatment. 5 Furthermore, up to 30% of previously healthy geriatric cats will develop CKD over the course of 12 months. 49 Such an occurrence could confound interpretation of results if follow-up periods after radioiodine treatment increase beyond 3 months.
Our study had some limitations. First, we were unable to measure GFR concurrently with other variables of renal and thyroid function.
Second, the study concentrated solely on measurement of serum TT4 concentration; a complete assessment of thyroid status was not performed. Hence, we could not define subpopulations with iatrogenic or subclinical hypothyroidism. Furthermore, the precise serum TT4 concentration was not known for cats with results >15 μg/dL