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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

Objectives

To evaluate calcium and phosphate homeostasis in hyperthyroid cats and determine if plasma parathyroid hormone and fibroblast growth factor-23 are associated with the presence of -azotaemic chronic kidney disease and/or have prognostic significance.

Methods

Retrospective cohort study. Logistic regression analysis and Cox regression analysis were performed to identify if parathyroid hormone and fibroblast growth factor-23 were predictors of development of azotaemia following treatment and survival time, respectively.

Results

Two hundred and seven hyperthyroid cats were included. Elevated plasma parathyroid hormone concentrations, hyperphosphataemia, decreased plasma fibroblast growth factor-23 concentrations and hypocalcaemia were documented; however, all parameters returned to reference intervals following treatment of hyperthyroid cats without azotaemic chronic kidney disease. After adjustment for plasma creatinine concentration, baseline plasma parathyroid hormone and fibroblast growth factor-23 concentrations were not predictors of the development of azotaemia following treatment. Baseline plasma fibroblast growth factor-23 concentrations were associated with all-cause mortality; however, this association was not maintained after adjustment for packed cell volume.

Clinical Significance

Changes in plasma parathyroid hormone and fibroblast growth factor-23 concentrations which occur in hyperthyroid cats are not mediators of progression of chronic kidney disease; however, fibroblast growth factor-23 would appear to have some prognostic significance in hyperthyroidism.

Hyperthyroidism and chronic kidney disease (CKD) are both common conditions of geriatric cats that can occur concurrently. CKD is reported to affect 31% of cats over 15 years of age (Lulich and others 1992), and 10% of cats with hyperthyroidism are reported to have concurrent azotaemic CKD at diagnosis (Williams and others 2010b). Hyperthyroidism complicates the diagnosis of concurrent CKD, because excess thyroid hormones cause an increase in glomerular filtration rate (GFR) and a -reduction in plasma creatinine concentrations (Adams and others 1997a). Consequently, renal function in hyperthyroid cats can only be fully assessed once hyperthyroidism is treated and euthyroidism is restored. It is reported that 15 to 49% of initially non-azotaemic hyperthyroid cats develop azotaemia within six months of successful treatment of hyperthyroidism (Graves and others 1994, Adams and others 1997b, Becker and others 2000, Slater and others 2001, Boag and others 2007, Riensche and -others 2008, Williams and others 2010b); therefore, the -prevalence of CKD in hyperthyroid cats would appear to be higher than the reported prevalence of CKD in the general geriatric population. For this reason, it could be postulated that damage occurs to the kidney in the hyperthyroid state, which increases the risk of developing azotaemic CKD.

Hyperthyroidism is positively associated with hyperphosphataemia and elevations in plasma parathyroid hormone (PTH) concentrations in cats (Barber and Elliott 1996). By contrast, human patients with Graves’ disease have decreased serum PTH concentrations in conjunction with elevated serum ionised calcium and serum phosphate concentrations (Mosekilde and others 1990, Iqbal and others 2003). The combination of elevated PTH concentrations and hyperphosphataemia is also observed in secondary renal hyperparathyroidism, and therefore could be present in hyperthyroid cats secondary to the presence of underlying CKD (Barber and Elliott 1996). Hence, it could be postulated that calcium and phosphate regulation in hyperthyroid cats differs markedly from that of human patients with Graves’ disease, because approximately 30% of hyperthyroid cats have concurrent CKD, whereas CKD is unlikely to be present in human patients as they are generally young to middle aged. Secondary renal hyperparathyroidism may contribute to the progression of CKD in cats, as the feeding of phosphate-restricted diets to cats with CKD has been shown to reduce PTH concentrations in cats with CKD (Barber and others 1999) and improve survival time (Elliott and others 2000). PTH is also regarded as a uraemic toxin in itself, because it has been associated with disorders of glucose and lipid metabolism, immunosuppression and defects in neurological function (Slatopolsky and others 1980), and PTH has been described as an independent marker of survival in human patients on haemodialysis (Avram and others 1996). As hyperthyroid cats have been demonstrated to have elevated PTH concentrations (Barber and Elliott 1996), hyperthyroid-associated elevations in plasma PTH concentrations could be one mechanism of renal damage or increased morbidity and hence mortality in feline hyperthyroidism.

Fibroblast growth factor-23 (FGF-23) is a recently discovered phosphatonin that is secreted by osteoblasts (Seiler and others 2009) in response to hyperphosphataemia and increased plasma concentrations of calcitriol (Tsagalis and others 2009). FGF-23 inhibits sodium-dependent phosphate reabsorption in the proximal tubule of the kidney thus increasing phosphaturia (Shimada and others 2004). FGF-23 is also postulated to have a central role in the development of secondary renal hyperparathyroidism, as FGF-23 inhibits the activity of renal 1α-hydroxylase leading to calcitriol deficiency (Seiler and others 2009). In human patients with Graves’ disease, serum FGF-23 concentrations are reported to be elevated and to decrease following treatment of hyperthyroidism (Yamashita and others 2005). Serum FGF-23 concentrations predict the progression of CKD in human patients (Fliser and others 2007), and have also been associated with all-cause mortality independent of other factors, such as serum PTH and phosphate concentrations, in one study of human haemodialysis patients (Gutierrez and others 2008). The biological significance of this observation however remains unclear, as FGF-23 could represent a biomarker of progressive disease, or alternatively FGF-23 could be a mediator of progression of renal disease. As FGF-23 is associated with the progression of CKD and might influence the development of hyperthyroid-associated elevations in plasma PTH concentrations, evaluation of plasma FGF-23 concentrations in hyperthyroid cats is warranted.

This study aimed to evaluate calcium and phosphate homeostasis in hyperthyroid cats, before and after treatment of cats with and without CKD, by measurement of routine plasma biochemical parameters and plasma PTH and FGF-23 concentrations. Baseline plasma PTH and FGF-23 concentrations were correlated with the presence of CKD and all-cause mortality to assess the prognostic significance of elevations in plasma PTH and FGF-23 concentrations in hyperthyroid cats. An additional aim was to validate an immunoradioimmetric assay (IRMA) for measurement of intact PTH in feline plasma.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

Patient selection

Records from two London-based first-opinion practices (People's Dispensary for Sick Animals, Bow and the Beaumont Sainsbury Animals’ Hospital, Camden) between January 1, 1999 and May 28, 2009 were reviewed and newly diagnosed hyperthyroid cats identified. Diagnosis of hyperthyroidism was based on a plasma total thyroxine concentration (TT4) greater than the laboratory reference interval (>55 nmol/L) or a failure of TT4 concentration to suppress to <20 nmol/L following administration of 20 µg liothyronine sodium (T3) three times daily for two days, then 20 µg liothyronine sodium (T3) on day 3, given 2 to 4 hours before sampling.

The clinical records were reviewed and the following data collected from the time of diagnosis: age, sex, systolic arterial blood pressure (SBP), plasma TT4 concentration, packed cell volume (PCV), routine plasma biochemical variables (total protein, albumin, globulin, urea, creatinine, total bilirubin, cholesterol, sodium, potassium, chloride, inorganic phosphorus, total calcium concentrations and activities of alanine aminotransferase and alkaline phosphatase), urine specific gravity (USG) and urine culture result (if available). The time to development of azotaemia (defined below) following treatment of hyperthyroidism was also documented. Cats were excluded from the analysis of factors associated with the development of azotaemia if they were azotaemic at, or before, diagnosis of hyperthyroidism.

Cats included in the study were treated for hyperthyroidism with antithyroid medication (carbimazole or methimazole) alone, or in combination with thyroidectomy.

A group of 28 healthy non-azotaemic (defined below) geriatric cats (older than nine years) were also recruited to the study. All cats were followed for a one-year period following sampling to ensure that azotaemia did not develop. Only cats which remained non-azotaemic during the one-year follow-up period were included in the healthy non-azotaemic group.

Blood pressure measurement

SBP measurements were made using an 8·1 MHz Doppler ultrasound probe following the protocol previously described (Syme and others 2002). Fundic examination by indirect -ophthalmoscopy was performed in cats with an average SBP greater than 160 mmHg to assess for evidence of hypertensive retinopathy. Hypertension was diagnosed if cats had an average SBP greater than 170 mmHg with evidence of hypertensive retinopathy, or if the average SBP was greater than 170 mmHg on two consecutive occasions, and the clinician did not feel that white coat hypertension was a significant contributing factor.

Categorisation of azotaemia and control of hyperthyroidism

Renal azotaemia was defined as a plasma creatinine concentration greater than 177 µmol/L in conjunction with inadequate urine concentrating ability (USG<1·035), or persistent azotaemia on two or more consecutive occasions without evidence of a prerenal cause.

Hyperthyroidism was categorised as well controlled if cats appeared euthyroid on clinical examination (no tachycardia or weight loss) and had serial TT4 measurements <40 nmol/L for a six-month period. This time point was used as the six-month post-control time point.

Blood and urine sampling and processing

Blood and urine samples were collected as part of a geriatric screening and healthcare programme at the time of diagnosis with the consent of the owner. Jugular venous blood samples were collected and placed in heparinised tubes, and urine samples collected by cystocentesis. Samples were kept at 4°C before sample processing which occurred within 6 hours of sample collection. Blood samples were placed in a centrifuge at 2016×g for 10 minutes to enable separation of plasma from cellular components. Heparinised plasma was submitted to a single external laboratory (Idexx Laboratories, Wetherby, UK) for biochemical analysis including TT4. Ethylenediaminetetraacetic acid (EDTA) plasma was stored at −80°C until batch analysis of PTH and FGF-23 which occurred between 2 and 12 years later. Samples used for measurement of plasma PTH concentration had not been thawed before analysis, and samples used for measurement of plasma FGF-23 concentrations were subjected to a maximum of one freeze-thaw cycle before measurement of FGF-23. Plasma FGF-23 concentrations were measured in EDTA plasma using a commercially available human intact FGF-23 ELISA (Human intact FGF-23 ELISA, Kainos Laboratories, Tokyo, Japan) that has been recently validated for use in feline plasma samples (Geddes and others 2011).

Urine samples underwent full in-house urinalysis including measurement of USG by refractometry, dipstick analysis and urine sediment examination. If bacteria or pyuria was identified on sediment examination then urine was submitted for bacterial culture and sensitivity. Urine samples that were positive on urine culture or which were grossly haematuric were excluded from UPC analysis. Urine samples were centrifuged at 2016×g for 10 minutes and the supernatant separated from any sediment. This was stored at −80°C until batch analysis of UPC. Urine protein concentration was measured by a colorimetric pyrogallol red method and urine creatinine measured by a colorimetric picric acid method.

Cats were re-examined at approximately eight-week (if treated with antithyroid medication) or three-month (if treated -surgically) intervals after diagnosis. Blood and urine samples were obtained approximately every three to four months.

Validation of PTH assay

A commercially available IRMA (total intact PTH IRMA-coated bead version, Part number 3KG600, Scantibodies, Santee, CA, USA) for measurement of human intact PTH was validated for use in feline plasma samples. To determine the dilutional parallelism, and hence the specificity of the assay, samples were diluted with the zero calibrator provided by the kit manufacturers to give dilutions of 1:2 and 1:4. The PTH concentrations at each dilution were then compared with expected concentrations calculated using the undiluted concentration measured by the assay. Precision and repeatability of the assay were assessed by evaluating intra- and inter-assay coefficients of variation for plasma samples with low and medium plasma PTH concentrations. The limit of detection was determined by measurement of the counts per minute (CPM) after incubation of the zero standard on five different assays, with the limit of detection being the interpolated PTH concentration of the mean CPM of zero standard+(3*standard deviation of CPM of zero standard) (Armbruster and Pry 2008).

Survival analysis

Survival times were calculated from the date of diagnosis of hyperthyroidism to the date of death or euthanasia. If these data were not available, the owner was contacted by telephone to request follow-up information. If the cat had died and the exact date of death was unknown, the month of death was recorded and it was assumed that the cat died on the 15th of that month for the purposes of the survival analysis. Cats were censored in the survival analysis if they were still alive at the end of the follow-up period (May 4, 2011) or if they were lost to follow-up. Cats were categorised as lost to follow-up if they had not attended the clinic for six months and their owners were not contactable by telephone.

Statistical analysis

Statistical analyses were performed using computerised statistical software (PASW version 18.0). Results are reported as median [25th, 75th percentile] and statistical significance was defined as P<0·05 unless otherwise stated. The Kruskal-Wallis test was used to compare median plasma PTH and FGF-23 concentrations before treatment between cats which had evidence of azotaemic CKD at the time of diagnosis, cats which developed azotaemia within 240 days of establishment of euthyroidism and cats which remained non-azotaemic after treatment. The Mann–Whitney U-test was used to make pairwise comparisons between the groups to identify significant differences. Bonferroni correction was applied to correct for multiple comparisons; therefore, statistical significance for this post hoc analysis was defined as P<0·017. The Wilcoxon signed rank test was used to compare plasma PTH and FGF-23 concentrations at the baseline and 6-month post-control time point in cats with and without azotaemic CKD.

Multivariable linear regression analysis was performed to identify independent predictors of plasma PTH and FGF-23 concentrations. Variables were logarithmically transformed as appropriate to ensure that the assumptions of linear regression analysis were satisfied. Baseline clinicopathological variables that were significantly associated (P<0·05) with plasma PTH and FGF-23 concentrations in univariable linear regression analysis were entered into multivariable models.

Plasma creatinine, PTH and FGF-23 concentrations were entered into a multivariable logistic regression model for the development of azotaemia within 240 days of establishment of euthyroidism. Plasma FGF-23 concentrations were divided by 100 and USG was multiplied by 1000 in order to allow easier interpretation of hazard and odds ratios. For the survival analysis, available continuous data for plasma PTH and FGF-23 concentrations were entered into a univariable Cox regression model to determine if they were associated with survival (P<0·2). Two survival models were constructed, the first model included age and plasma concentrations of creatinine, total calcium, phosphate, PTH and FGF-23, and the second model also included other variables previously associated with all-cause mortality (Williams and others 2010b). For each model, the variables were included in backward, stepwise multivariable Cox regression analyses for survival. Available continuous data were checked to ensure that the data met the assumption of proportional hazards. Data were divided into terciles if this assumption was not met, and first-order interactions were assessed.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

The intact PTH radioimmunoassay demonstrated adequate dilutional parallelism with (mean ±sd) 102 ±11% and 104 ±17% (n=5) of expected concentrations at dilutions of 1:2 and 1:4, respectively. The intra-assay variability of samples with low (8·7 pg/mL) and medium (33·1 pg/mL) plasma concentrations of PTH were 18·9% and 11%, respectively. The inter-assay variability for samples with low and medium plasma PTH concentrations were 12·3% and 11·9%, respectively. The limit of detection was calculated to be 5·2 pg/mL.

Plasma PTH concentrations were measured in 28 non-azotaemic cats and ranged between <5·2 and 17·3 pg/mL. Plasma PTH concentrations were measured in 200 hyperthyroid cats at baseline and ranged between <5·2 and 284·5 pg/mL (Fig 1). One hundred and fourteen hyperthyroid cats (57%) had a plasma PTH concentration greater than 17·3 pg/mL. Hyperthyroid cats which had evident azotaemic CKD at diagnosis had a plasma PTH concentration of 26·5 [17·4, 61·8] pg/mL (n=17, Fig 1), of which 14 (82%) had a plasma PTH concentration greater than 17·3 pg/mL. Hyperthyroid cats which developed azotaemia within 240 days of establishment of euthyroidism had a plasma PTH concentration of 21·5 [14·2, 48·8] pg/mL (n=35, Fig 1), of which 24 (69%) had a plasma PTH concentration greater than 17·3 pg/mL. Cats which remained non-azotaemic after treatment had a plasma PTH concentration of 18·8 [11·2, 33·3] pg/mL (n=148, Fig 1), of which 77 (52%) had a plasma PTH concentration greater than 17·3 pg/mL. Plasma PTH concentrations were not significantly different between the three groups at baseline (P=0·067).

image

Figure 1. Plasma parathyroid hormone (PTH) concentrations of 200 hyperthyroid cats at diagnosis. Cats were divided into three groups: azotaemic – hyperthyroid cats which were azotaemic at, or before, the time of diagnosis (n=17); pre-azotaemic – hyperthyroid cats which developed azotaemia within 240 days of establishment of euthyroidism (n=35); and non-azotaemic – cats which remained non-azotaemic for at least 240 days following establishment of euthyroidism (n=148) and compared using the Kruskal-Wallis test. The dotted line represents the upper limit of the normal reference interval for geriatric cats derived in this study (17·3 pg/mL). There were no significant differences between the three groups at baseline (P=0.067).

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Plasma PTH concentrations decreased following treatment of cats which remained non-azotaemic, whereas plasma PTH concentrations did not change significantly following treatment in cats which developed azotaemia following treatment (Table 1).

Table 1. Selected clinicopathological variables before and after treatment of hyperthyroidism in cats
VariablePretreatment median[25th, 75th percentiles]Post-treatment median[25th, 75th percentiles]nSig.
  1. Sig. Significance, PTH Plasma parathyroid hormone, FGF Fibroblast growth factor. Cats were divided into two groups: azotaemic post-treatment group – cats which develop azotaemia within 240 days of establishment of euthyroidism; non-azotaemic – cats remained non-azotaemic for a 240-day period following establishment of euthyroidism. The Wilcoxon signed rank test was used to compare selected variables before and after treatment of hyperthyroidism.

Plasma creatinine concentration (µmol/L)    
Azotaemic post-treatment109·9 [92·3, 133·4]193·6 [187·5, 263·3]17<0·001
Non-azotaemic97·4 [81·9, 115·4]141·9 [121·3, 162·1]28<0·001
Plasma phosphate concentration (mmol/L)    
Azotaemic post-treatment1·51 [1·23, 1·95]1·31 [1·04, 1·77]170·309
Non-azotaemic1·55 [1·34, 1·76]1·27 [1·11, 1·41]28<0·001
Plasma total calcium concentration (mmol/L)    
Azotaemic post-treatment2·47 [2·34, 2·52]2·43 [2·26, 2·53]160·313
Non-azotaemic2·41 [2·32, 2·52]2·47 [2·36, 2·58]260·003
Plasma PTH concentration (pg/mL)    
Azotaemic post-treatment19·5 [16·3, 58·3]26·6 [11·5, 55·4]100·646
Non-azotaemic13·8 [9·5, 20·0]9·7 [7·7, 14·8]130·023
Plasma FGF-23 concentration (pg/mL)    
Azotemic post-treatment218 [176, 372]516 [259, 1621]110·008
Non-azotaemic165 [129, 261]215 [163, 308]220·013

Plasma FGF-23 concentrations were measured in 207 hyperthyroid cats at baseline and ranged between 23 and 8859 pg/mL (Fig 2). One previous study reported that plasma FGF-23 concentrations in 15 healthy non-azotaemic cats ranged between 69 and 505 pg/mL (Finch and others 2011). Hyperthyroid cats which had evident azotaemic CKD at the time of diagnosis had a plasma FGF-23 concentration of 741 [394, 2298] pg/mL (n=16, Fig 2). Hyperthyroid cats which developed azotaemia within 240 days of establishment of euthyroidism had a plasma FGF-23 concentration of 298 [186, 463] pg/mL (n=37, Fig 2) and cats which remained non-azotaemic after treatment had a plasma FGF-23 concentration of 216 [146, 336] pg/mL (n=154, Fig 2). There was a significant difference in plasma FGF-23 among all three groups (P<0·017 for all pairwise comparisons).

image

Figure 2. Plasma fibroblast growth factor-23 (FGF-23) concentrations of 207 hyperthyroid cats at diagnosis. Cats were divided into three groups: azotaemic – hyperthyroid cats which were azotaemic at, or before, the time of diagnosis (n=16); pre-azotaemic – hyperthyroid cats which developed azotaemia within 240 days of establishment of euthyroidism (n=37); and non-azotaemic – cats which remained non-azotaemic for at least 240 days following establishment of euthyroidism (n=154) and compared using the Kruskal-Wallis test. The Mann–Whitney test with Bonferonni correction was used to make pairwise comparisons with significance defined as P<0·017. The dotted lines represent the upper and lower limits of the normal reference interval derived in a previous study (69 to 505 pg/mL). Plasma FGF-23 concentrations were significantly different between all three groups (P<0.017 for all pairwise comparisons).

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Plasma FGF-23 concentrations increased following treatment in both cats which remained non-azotaemic and cats which developed azotaemia following treatment (Table 1).

Plasma phosphate concentration decreased following treatment in cats which remained non-azotaemic, whereas plasma phosphate did not change significantly following treatment in cats which developed azotaemia (Table 1). Conversely, plasma total calcium concentration increased following treatment of non-azotaemic cats, whereas it did not change following treatment of azotaemic cats (Table 1).

Linear regression analysis identified PCV, SBP and plasma concentrations of TT4, creatinine, total calcium, phosphate and FGF-23 as being significantly associated with log plasma PTH concentrations; however, only plasma phosphate (P<0·001) and plasma creatinine (P=0·041) concentrations remained significant predictors of log plasma PTH concentration in the multivariable model (adjusted r2=0·368, n=154; P<0·001; Table 2).

Table 2. Multivariable linear regression model of predictors of log plasma PTH concentration in hyperthyroid cats at -diagnosis (adjusted r2=0·368, n=154; P<0·001)
VariableBseSig.95% CI for B
  1. PTH Plasma parathyroid hormone, se Standard error, Sig. Significance, CI Confidence interval, FGF-23 Fibroblast growth factor-23.

Packed cell volume (%)−0·0080·0040·055−0·016 to 0·000
Systolic blood pressure (mmHg)0·0010·0010·314−0·001 to 0·003
Plasma total thyroxine concentration (nmol/L)0·0010·0000·0830·000 to 0·002
Plasma creatinine concentration (µmol/L)0·0020·0010·0410·000 to 0·003
Plasma total calcium concentration (mmol/L)−0·2170·1470·142−0·507 to 0·073
Plasma phosphate concentration (mmol/L)0·2650·054<0·0010·159 to 0·371
Plasma FGF-23 concentration (pg/mL)/1000·0010·0020·536−0·003 to 0·005
Constant1·2260·3580·0010·519 to 1·933

Linear regression analysis identified PCV, and plasma concentrations of creatinine, total calcium, phosphate and PTH as being significantly associated with log plasma FGF-23 concentrations, and all five variables remained associated with log plasma FGF-23 concentration in the multivariable linear regression model (adjusted r2=0·410, n=156; P<0·001; Table 3).

Table 3. Multivariable linear regression model of predictors of log plasma FGF-23 concentration in hyperthyroid cats at diagnosis (adjusted r2=0·410, n=156, P<0·001)
VariableiseSig.95% CI for B
  1. FGF-23 Fibroblast growth factor-23, se Standard error, Sig. Significance, CI Confidence interval, PTH Plasma parathyroid hormone.

Packed cell volume (%)−0·0150·0050·002−0·024 to 0·006
Plasma creatinine -concentration (µmol/L)0·0030·001<0·0010·001 to 0·004
Plasma total -calcium -concentration (mmol/L)0·4020·170·0190·066 to 0·738
Plasma phosphate -concentration (mmol/L)0·1540·0630·0150·030 to 0·279
Plasma PTH concentration (pg/mL)0·0030·0010·0050·001 to 0·005
Constant1·3450·3980·0010·559 to 2·130

One hundred and fifty-four cats were included in the logistic regression analysis, of which 30 developed azotaemia within 240 days of establishment of euthyroidism. Logistic regression analysis revealed that plasma PTH and FGF-23 concentrations were not predictors of the development of azotaemia following treatment after adjustment for plasma creatinine concentration; however, plasma creatinine concentration was a significant predictor of the development of azotaemia after adjustment for plasma PTH and FGF-23 concentrations (Table 4). The Hosmer-Lemenshow test confirmed good model fit (P=0·385).

Table 4. Multivariable logistic regression model of factors associated with the development of azotemia following -treatment of hyperthyroidism (n=154)
Explanatory variableBseSig.Odds ratio95% CI for OR
  1. B Coefficient, se Standard error, Sig. Significance, OR Odds ratio, CI Confidence interval, PTH Plasma parathyroid hormone, FGF-23 Fibroblast growth factor-23.

  2. Plasma FGF-23 concentrations were divided by 100 in order to make the odds ratio easier to interpret. The odds ratio indicates the increase in the odds of developing azotemia for each unit increase in the explanatory variable.

Creatinine (µmol/L)0·0360·009<0·0011·0361·018 to 1·054
PTH (pg/mL)−0·0010·0070·8940·9990·985 to 1·013
FGF-23/100 (pg/mL)−0·0100·0280·7210·9900·928 to 1·048
Constant−1·3531·4530·3520·259 

Univariable Cox regression analysis indicated that both plasma PTH [hazard ratio (HR)=1·006, 95% CI for HR=1·002 to 1·009, n=198; P=0·001] and FGF-23 concentrations (HR for FGF-23 concentration/100=1·026, 95% CI for HR=1·016 to 1·036, n=203; P<0·001) were negatively associated with survival time and thus eligible for inclusion in the multivariable models. The first survival model included 152 hyperthyroid cats, of which 24 (16%) were censored from the analysis as they were still alive or had been lost to follow-up at the end of the study. In the final model (Table 5, Fig 3), survival was negatively -associated with both age (P<0·001) and plasma FGF-23 concentration (P<0·001; Table 5). The second survival model included 93 hyperthyroid cats of which 12 (13%) were censored from the analysis. In the final model (Table 6), survival was positively correlated with PCV (P=0·006) and negatively correlated with the presence of hypertension at the time of diagnosis of hyperthyroidism (P=0·039) and UPC (P=0·041); however, plasma FGF-23 concentration was no longer associated with survival time. First-order interactions were assessed in all models and no significant (P<0·05) interactions were identified.

Table 5. Backward stepwise Cox regression multivariable model of factors associated with survival in hyperthyroid cats at diagnosis (n=152)
VariablenBseSig.HR95% CI for HR
  1. n Number of cats in group, B Coefficient, se Standard error, Sig. Significance, HR hazard ratio, CI Confidence interval, FGF fibroblast growth factor-23

  2. Age and plasma concentrations of creatinine, total calcium, phosphate, FGF-23 and parathyroid hormone were entered into the initial model. The hazard ratio represents the effect of a unit change in the predictor variable on the frequency of the outcome (death) (Dohoo and others 2003).

Age (years)1520·1020·0310·0011·1071·042 to 1·176
Plasma FGF-23 (pg/mL)      
  ≤18251  <0·001  
  183 to 33447−0·0100·2300·9660·9900·631 to 1·555
  >334540·7660·2210·0012·1521·396 to 3·317
image

Figure 3. Survival curves constructed using a backward, stepwise multivariable Cox regression analysis with stratification according to plasma fibroblast growth factor-23 (FGF-23) concentration in 152 hyperthyroid cats. The analysis was controlled for age and plasma concentrations of creatinine, phosphate, total calcium and parathyroid hormone. The blue and green curves are superimposed as these two groups have hazard ratios that are similar (see Table 5)

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Table 6. Backward stepwise Cox regression multivariable model of factors associated with survival in hyperthyroid cats at diagnosis (n=93)
VariablenBseSig.HR95% CI for HR
  1. n Number of cats in group, B Coefficient, se Standard error, Sig. Significance, HR Hazards ratio, CI Confidence interval, PCV Packed cell volume, USG Urine specific gravity, UPC Urine protein:creatinine ratio.

  2. Age, hypertensive status at diagnosis, PCV, USG (×1000), UPC, and plasma concentrations of creatinine, total calcium, phosphate, fibroblast growth factor-23 and parathyroid hormone were entered into the initial model. The hazard ratio represents the effect of a unit change in the predictor variable on the frequency of the outcome (death) (Dohoo and others 2003).

Hypertensive status

Normotensive

Hypertensive

76

17

0·6880·3330·0391·9891·035 to 3·824
PCV (%)

≤35

36 to 39

>39

39

26

28

−0·845

−0·936

0·314

0·334

0·006

0·007

0·005

0·430

0·392

0·232 to 0·795

0·204 to 0·755

USG (×1000)93−0·0180·0090·0550·9830·965 to 1·000
UPC

≤0·36

0·37 to 0·73

>0·73

29

35

29

0·568

0·849

0·320

0·344

0·041

0·076

0·014

1·764

2·337

0·942 to 3·302

1·190 to 4·588

Plasma creatinine (µmol/L)

≤90·8

90·9 to 117·8

>117·8

32

27

34

−0·317

0·468

0·320

0·406

0·089

0·323

0·249

0·729

1·598

0·389 to 1·365

0·721 to 3·542

Plasma phosphate (mmol/L)

≤1·40

1·41 to 1·79

>1·79

30

26

37

0·640

−0·122

0·357

0·296

0·086

0·073

0·682

1·896

0·886

0·941 to 3·820

0·495 to 1·583

Plasma urea and creatinine concentrations were not included in the same multivariable models as the two variables were highly correlated. Substitution of plasma urea into the models resulted in plasma FGF-23 concentration no longer being associated with survival time in any model (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

The intact PTH IRMA validated in the present study demonstrated good precision, reproducibility and accuracy. Healthy non-azotaemic cats had plasma PTH concentrations ranging between <5·2 and 17·3 pg/mL; however, this range also encompassed the lower limit of detection of the assay. The number of cats included in the present study was not sufficient to derive a reference interval; therefore, further studies are required to evaluate PTH concentrations in healthy geriatric cats using this assay.

PTH is degraded to form various N- and C-terminal fragments within minutes of secretion by the parathyroid gland (Komaba and others 2009), and C-terminal fragments of PTH are known to accumulate in human patients with CKD (Nguyen-Yamamoto and others 2002). The manufacturers of the intact PTH kit used in the present study report almost 100% cross-reactivity with the large C-terminal fragments with partially preserved N-terminal structure [PTH(7–84)]; however, the assay has minimal cross-reactivity with smaller C-terminal fragments, the detection of which made older PTH assays less reliable in patients with renal dysfunction. Use of whole PTH assays which only measure the complete PTH molecule [PTH(1–84)] would avoid measurement of C-terminal fragments that are reported to accumulate in CKD; however, other PTH fragments with preserved structural integrity of the PTH(1–4) region have recently been identified which would be cross-reactive with whole PTH assays but not with intact PTH assays (D'Amour and others 2003). One recent review concluded that there is currently no evidence that intact PTH assays give poorer information than whole PTH assays in human patients (Souberbielle and others 2010). In the present study, plasma PTH concentration did not differ between hyperthyroid cats with and without CKD at -baseline; therefore, this would suggest that the elevated plasma PTH concentrations observed in cats with hyperthyroidism were not only reflective of reduced renal function and the accumulation of large C-terminal fragments. The results of linear regression analysis indicated that plasma PTH concentration is independently correlated with plasma phosphate concentration and plasma creatinine concentration. These results would be expected, as hyperphosphataemia will directly stimulate the parathyroid gland to secrete PTH (Slatopolsky and others 1996), and increased plasma creatinine concentration will reflect reduced GFR resulting in increased accumulation of C-terminal PTH fragments and whole PTH that will be detected by the intact PTH assay. As hypocalcaemia also stimulates PTH secretion by the parathyroid gland (Naveh-Many and others 1989), it would be expected that plasma calcium concentration would be associated with plasma PTH concentration; however, the model used plasma total calcium concentration as a predictive variable rather than ionised calcium concentration. One study recently showed that plasma total calcium and ionised calcium concentration in cats were poorly -correlated (Schenck and Chew 2010). The model only explained 37% of the variability in plasma PTH concentration; therefore, a model which incorporated ionised calcium concentration as a predictive variable might have indicated that ionised calcium was a predictor of PTH concentration and might have explained a greater proportion of the variability in PTH.

The results of the present study suggest that approximately 60% of hyperthyroid cats have elevated plasma PTH concentrations, which is similar to the findings of a previous study (Barber and Elliott 1996). It was postulated that the elevated plasma PTH concentrations that were seen in hyperthyroid cats could be due to secondary renal hyperparathyroidism that was present in hyperthyroid cats with underlying CKD. It is known that 15 to 49% of initially non-azotaemic hyperthyroid cats develop -azotaemia within 6 months of successful treatment of hyperthyroidism (Graves and others 1994, Adams and others 1997b, Becker and others 2000, Slater and others 2001, Boag and others 2007, Riensche and others 2008, Williams and others 2010b), and 84% of cats with azotaemic CKD have secondary renal -hyperparathyroidism (Barber and Elliott 1998). In the present study, 69% of cats which developed azotaemia following treatment had plasma PTH concentrations greater than 17·3 pg/mL; however, 55% of cats which remained non-azotaemic following treatment also had elevated plasma PTH concentrations. It has been reported that cats in the pre-azotaemic stages of CKD can have elevated plasma PTH concentrations (Finch and others 2009); however, it would seem unlikely that 55% of the non-azotaemic group in the present study were in the pre-azotaemic stages of CKD. The results of the longitudinal analysis also suggest that plasma PTH concentrations decrease following treatment in hyperthyroid cats which remain non-azotaemic and remain elevated in cats which develop azotaemia. The elevated post-treatment plasma PTH concentration in cats with azotaemia probably reflects the presence of secondary renal hyperparathyroidism which is reported to be present in 84% of cats with azotaemic CKD (Barber and Elliott 1996). These findings also support the hypothesis that hyperthyroidism causes elevated PTH concentrations through a pathophysiological mechanism other than secondary renal hyperparathyroidism.

The mechanism for the development of hyperthyroid-associated elevations in plasma PTH concentrations is unknown; however, the results of the present study show that hyperthyroid cats without underlying azotaemic CKD have decreased plasma total calcium concentrations and increased plasma phosphate concentrations. These findings are again in agreement with a previous study (Barber and Elliott 1996) which reported that 27% of hyperthyroid cats had an ionised hypocalcaemia and 43% of hyperthyroid cats were hyperphosphataemic. Another study reported that 50% of hyperthyroid cats had ionised hypocalcaemia (Archer and Taylor 1996). Hypocalcaemia and hyperphosphataemia would be expected to stimulate PTH secretion by the parathyroid gland (Naveh-Many and others 1989, Slatopolsky and others 1996).

The cause of hyperphosphataemia in hyperthyroid cats is currently unknown. Hyperphosphataemia could result from increased phosphate absorption in the gut, increased renal reabsorption in the kidney or increased release of phosphate secondary to bone resorption. Increased bone turnover in feline hyperthyroidism has been suggested in two studies; the first study reported increased plasma osteocalcin concentrations (Archer and Taylor 1996), and the second preliminary study reported increased osteocalcin, bone-derived ALP, deoxypyridinoline, carboxy-terminal propeptide of type I collagen, carboxy-terminal telopeptide of type I collagen and serum cross-linked carboxy-terminal collagen telopeptide in hyperthyroid cats all of which normalised following treatment with radioiodine (Slater and others 2004). However, increased bone turnover would be expected to release calcium as well as phosphate, thus resulting in hypercalcaemia, which does not appear to occur in cats with hyperthyroidism.

Thyroid hormones are known to act on the proximal tubular cells of the kidney to cause increased phosphate reabsorption (Yusufi and others 1985), and studies in rats have demonstrated that physiological doses of thyroid hormone can stimulate renal phosphate reabsorption resulting in an increased serum phosphate concentration (Alcalde and others 1999). If this were to also occur in hyperthyroid cats, this mechanism could account for the development of hyperphosphataemia without concurrent hypercalcaemia.

The reason why hyperthyroid cats have ionised hypocalcaemia is also unknown, although it could be a secondary effect of hyperphosphataemia in hyperthyroidism, as phosphate will complex with calcium causing a reciprocal decrease in ionised calcium concentration. In contrast, human Graves’ disease patients are reported to have hyperphosphataemia and hypercalcaemia (Yamashita and others 2005), which is suggested to be due to increased bone turnover in hyperthyroid patients (Mosekilde and others 1990). However, as cats also appear to have increased bone turnover, based on measurements of plasma markers of bone turnover (Archer and Taylor 1996), it might be expected that hyperthyroid cats should also have hypercalcaemia. One recent study of human Graves’ disease patients reported that there was increased serum parathyroid hormone-related peptide (PTH-rp) in human patients with Graves’ disease and that elevated PTH-rp could be involved in the pathogenesis of hypercalcaemia in hyperthyroidism (Giovanella and others 2011). The cause of increased PTH-rp secretion in Graves’ disease is not currently understood; however, one study demonstrated that experimental hyperthyroidism in rats caused increased expression of PTH-rp in the ventricular myocardium (Halapas and others 2008). Further work is warranted to determine if altered PTH-rp secretion is present in feline hyperthyroidism and to document the ionised calcium concentrations in a larger population of cats before and after treatment.

FGF-23 concentrations in hyperthyroid cats demonstrated to have azotaemic CKD at diagnosis or which develop it after treatment were significantly higher than plasma FGF-23 concentrations in non-azotaemic cats. As FGF-23 is secreted in response to hyperphosphataemia (Tsagalis and others 2009), and cats with CKD are reported to have increased plasma phosphate concentrations (Barber and Elliott 1996), the increased FGF-23 concentrations would be an expected and appropriate physiological response.

Unexpectedly, plasma FGF-23 concentrations increased following treatment of hyperthyroidism in cats with and without azotaemic CKD, suggesting that plasma FGF-23 concentrations are suppressed in feline hyperthyroidism. This is in contrast to human patients with Graves’ disease in which serum FGF-23 concentrations are reported to be elevated at diagnosis and to decrease following treatment of hyperthyroidism (Yamashita and others 2005). Plasma phosphate concentrations tended to decrease with treatment for hyperthyroidism, although this only reached significance in the non-azotaemic group. This would suggest that changes in plasma FGF-23 concentration that occur with treatment of hyperthyroidism are independent of changes in plasma phosphate concentration. It has been reported that GFR is the most important predictor of serum FGF-23 concentration in human CKD patients (Filler and others 2011), as FGF-23 is a low-molecular-weight protein which is freely filtered at the glomerulus and thus accumulates as GFR decreases. Treatment of hyperthyroidism causes a reduction in GFR (Adams and others 1997b, Boag and others 2007), and therefore it is possible that the increase in plasma FGF-23 concentration that occurs following treatment is secondary to the reduction in GFR. Changes in calcitriol concentrations might also have influenced FGF-23 concentrations; however, measurements of plasma calcitriol concentration were not available in the present study.

In multivariable linear regression, plasma FGF-23 was correlated with plasma concentrations of creatinine, phosphate, calcium, PTH and also PCV. These results would suggest that increasing plasma creatinine concentration (and hence reduced GFR) was correlated with increased plasma FGF-23 concentration, after adjustment for other factors, supporting the hypothesis that plasma FGF-23 concentrations were influenced by GFR. Plasma total calcium concentration was also correlated with plasma FGF-23 concentration in the multivariable linear regression model, suggesting a role of calcium in the control of FGF-23 secretion. One study reported that increased dietary intake of calcium stimulated FGF-23 secretion by bone (Shimada and others 2005), and in another study of patients with primary hyperparathyroidism, serum calcium concentration was an independent predictor of serum FGF-23 concentration (Yamashita and others 2004). Plasma PTH concentration was correlated with plasma FGF-23 concentration, after adjustment for other factors, suggesting that plasma PTH can have a direct effect on FGF-23 secretion, as has been suggested previously (Silver and Naveh-Many 2010). PCV was also negatively correlated with plasma FGF-23 concentrations in the present study, following adjustment for other factors. A relationship between PCV and FGF-23 has not been reported previously and might suggest an effect of FGF-23 on PCV, or vice versa.

FGF-23 has been associated with progression of CKD in human patients independent of other factors such as plasma phosphate and PTH concentrations (Fliser and others 2007). In cats, both plasma FGF-23 and PTH concentrations have been associated with the progression of CKD in cats in the pre-azotaemic stages of CKD (Finch and others 2009, 2011); however, the significance of these findings is uncertain, as PTH and FGF-23 could be markers or mediators of the progression of CKD. Secondary renal hyperparathyroidism may contribute to the progression of CKD in cats, as the feeding of phosphate-restricted diets to cats with CKD has been shown to reduce the PTH concentrations in cats with CKD (Barber and others 1999) and improve survival time (Elliott and others 2000). However, multivariable logistic regression analysis indicated that plasma PTH and FGF-23 concentrations were not associated with the development of azotaemia following treatment of hyperthyroidism following adjustment for other markers of renal function (plasma creatinine concentration). This suggests that the significant difference in baseline plasma FGF-23 concentration that was observed between hyperthyroid cats which remained non-azotaemic and those which developed azotaemia following treatment was reflective of reduced renal function and possibly increased accumulation of FGF-23 secondary to reduced GFR. Hence, it could be concluded that PTH and FGF-23 are not mediators of the progression of CKD in hyperthyroid cats.

FGF-23 is also independently associated with survival time in human CKD patients on haemodialysis independent of other baseline factors such as serum phosphate and PTH (Gutierrez and others 2008), and earlier studies also reported that PTH was a marker of survival (Avram and others 1996, Tentori and others 2008). As hyperthyroid cats have been demonstrated to have elevated PTH concentrations (Barber and Elliott 1996), hyperthyroid-associated elevations in plasma PTH concentrations could cause increased morbidity and hence mortality in feline hyperthyroidism. In the present study, only plasma FGF-23 concentration remained significantly associated with all-cause mortality following adjustment for age and plasma concentrations of creatinine, total calcium, phosphate and PTH. When plasma FGF-23 concentration was further adjusted for other factors that had previously been associated with all-cause mortality in hyperthyroid cats (Williams and others 2010b), plasma FGF-23 concentration was no longer associated with all-cause mortality. Interestingly, removal of PCV as an explanatory variable in the final model did result in plasma FGF-23 concentration remaining associated with survival time together with age, USG and UPC (data not shown). As PCV was negatively correlated with plasma FGF-23 concentration, it could be possible that FGF-23 influences survival time by causing reductions in PCV which in turn cause increased morbidity (and hence mortality) in cats with hyperthyroidism. Further work to determine the relationship between FGF-23 and PCV therefore might be justified to explore this relationship further.

Incorporation of plasma urea concentration into the models instead of plasma creatinine concentration resulted in plasma FGF-23 concentration no longer being associated with survival time. This might indicate that FGF-23 is not associated with survival time independent of renal function if plasma urea concentration is a superior marker of renal function in the hyperthyroid state. Plasma creatinine concentration is known to be decreased in hyperthyroidism as a result of increased GFR and decreased muscle mass, whereas plasma urea concentration should not be affected by changes in muscle mass. Therefore, it could be argued that the relationship between plasma FGF-23 concentration and survival time is reflective of the presence of underlying CKD. It is known that hyperthyroid cats with evident CKD at the time of diagnosis have shorter survival times than non-azotaemic hyperthyroid cats (Milner and others 2006, Williams and others 2010b); however, hyperthyroid cats which develop azotaemia following treatment (and thus which have underlying CKD) do not have shorter survival times compared to cats which remain non-azotaemic, provided that iatrogenic hypothyroidism is avoided (Williams and others 2010a). Therefore, the association between FGF-23 and survival time in hyperthyroid cats may not be explained entirely by the presence of concurrent CKD.

This study aimed to evaluate calcium and phosphate homeostasis in hyperthyroid cats with variable renal function before and after treatment; however, the retrospective nature of the study meant that some factors could not be included in the analysis. Measurement of plasma ionised calcium, calcitriol and PTH-rp concentrations were not possible, partly due to the limited number of stored samples available. Prospective studies would be required to investigate the changes that occur in these variables following treatment of hyperthyroidism, and to determine their roles in calcium and phosphate homeostasis in the hyperthyroid state.

One further potential limitation of this study was that the measurement of plasma PTH and FGF-23 concentrations did not occur until 2 to 12 years following sampling. If large amounts of degradation were to occur in samples kept at −80°C over many years, then it could be postulated that the duration of sample storage might affect the measured plasma PTH and -FGF-23 -concentrations. Several studies have investigated the effect of short-term storage of samples at −80°C on the measured plasma PTH and FGF-23 concentrations. First, a previous paper which validated another (now commercially unavailable) IRMA for the measurement of PTH in feline plasma reported that there was no significant difference in plasma PTH concentration following storage of samples at −80°C for a mean time of 50 days (Barber and others 1993). In addition, there would appear to be no significant difference in the measured plasma FGF-23 concentrations following storage of plasma at −20°C for a two-week period or following three freeze-thaw cycles (Finch 2011). Furthermore, in the present study, plasma PTH concentrations decreased in non-azotaemic hyperthyroid cats following treatment, and so plasma PTH concentrations were higher before treatment despite these samples being in storage for an additional six-month period. Another retrospective study carried out over a similar time period to the present study has also reported that plasma FGF-23 concentrations decrease following institution of renal diet for a four- to eight-week period in non-hyperthyroid azotaemic cats (Geddes and others 2012). If FGF-23 were degraded significantly during storage at −80°C, it would be expected that FGF-23 concentrations would be higher following institution of renal diet as these samples would have been stored for a shorter period of time. Hence, there would appear to be good evidence that the freezing of samples for short periods does not significantly influence the measured plasma PTH and FGF-23 concentrations.

The effect of long-term storage on the measured plasma PTH and FGF-23 concentrations is, however, uncertain. The manufacturers of the human intact FGF-23 ELISA state that FGF-23 remains stable in plasma stored at −80°C for three years; however, the effect of storage for longer periods is not discussed. Ideally, plasma PTH and FGF-23 would be measured in the same sample before and after long-term storage in order to ascertain if there is a storage effect; however, this is not practical. In the current study, there was no evidence of a negative correlation between storage time and plasma concentrations of PTH and FGF-23 (data not shown); therefore, the authors believe that it is unlikely that degradation of PTH and FGF-23 is a significant factor; however, further studies in companion animals are required to determine how much degradation of PTH and FGF-23 occurs during long-term storage of samples at −80°C.

In summary, hyperthyroidism appears to result in disruption of normal calcium and phosphate homeostasis, with hyperthyroid cats demonstrating elevated plasma PTH and phosphate concentrations and suppressed plasma FGF-23 and total calcium concentrations. The effects of hyperthyroidism on calcium and phosphate homeostasis are further complicated by the presence of concurrent CKD. The cause of the hyperphosphataemia and elevated plasma PTH concentrations that are associated with hyperthyroidism is uncertain; however, secondary renal hyperparathyroidism would not appear to be the sole pathophysiological mechanism. PTH and FGF-23 were not independently associated with the presence and progression of CKD in feline hyperthyroidism, and although FGF-23 was associated with all-cause mortality, this did not appear to be independent of changes in PCV.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

The authors would like to acknowledge BSAVA Petsavers who funded this study.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

None of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References
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