All work was performed at the Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. This work was funded by a BOF grant from Ghent University, Belgium. Results of this study were partially presented at the annual ACVIM forum in San Antonio, 2008.
Corresponding author: Ingrid van Hoek, DVM, PhD, Royal Canin S.A., Centre de Recherche, Aimargues, France; e-mail: email@example.com.
Background: Retinol-binding protein (RBP) is suggested as a clinically useful marker of renal function in cats.
Hypothesis: Serum and urinary RBP concentrations in hyperthyroid (HT) cats differ from those in healthy (H) cats; radioiodine (131I) treatment influences serum and urinary RBP concentrations in HT cats.
Animals: Ten HT and 8 H cats.
Methods: RBP concentration was evaluated in feline serum and urine samples from a prospective study.
Results: There was a significant (P= .003) difference in the urinary RBP/creatinine (uRBP/c) ratios of H (−) and untreated HT (1.4 ± 1.5 × 10−2 μg/mg) cats. Serum total thyroxine concentration (1.8 ± 1.9 μg/dL, 24 weeks) and uRBP/c (0.6 ± 1.0 × 10−2 μg/mg, 24 weeks) decreased significantly (P < .001) in HT cats at all time points after treatment with 131I, and these variables were significantly correlated with one another (r= 0.42, P= .007). Serum RBP concentrations from HT cats (199 ± 86 μg/L) did not differ significantly (P= .98) from those of H cats (174 ± 60) and did not change after treatment with 131I (182 ± 124 μg/L, P= .80).
Conclusion and Clinical Importance: The presence of urinary RBP in HT cats is a potential marker of tubular dysfunction that is correlated to thyroid status, although it is independent of circulating RBP concentrations. The decreased uRBP/c combined with the absence of changes in serum RBP after treatment suggests that the suspected tubular dysfunction was partly reversible with treatment of 131I.
Retinol binding protein (RBP) in cats is designated serum RBP or urinary RBP (uRBP). Serum RBP forms a holo-RBP complex with its retinol ligand,1–4 and holo-RBP binds to transthyretin, which prevents the loss of both serum RBP and its bound retinol through glomerular filtration.5 Only molecules with a molecular weight smaller than albumin (66 kDa) can pass through the glomerular barrier. RBP is a low molecular weight carrier protein of 21 kDa and tetrameric transthyretin has a molecular weight of 55 kDa. Upon release of its ligand, the uncomplexed apo-RBP no longer has affinity for transthyretin and can freely pass through the glomerular barrier and be reabsorbed through megalin receptor-dependent endocytosis in the proximal tubules.6 However, when tubular function fails, elimination of uRBP shifts from intratubular catabolism to urinary excretion.7 This tubular type of proteinuria is a highly sensitive index of renal tubular damage in humans because a minor decrease in tubular function can lead to the excretion of RBP in urine.8,9
RBP found in urine is modified from RBP in serum by proteolysis at the carboxyl terminus. Urinary RBP in cats reacts with antihuman (α-Hu) uRBP antibodies (Abs),10 and despite differences in the amino acid composition between serum RBP and uRBP, radial immunodiffusion techniques have shown partial but substantial cross-reactivity between feline serum and rabbit α-Hu uRBP Abs.10 Western analysis has confirmed the presence of RBP in feline plasma, liver, and kidney samples based on cross-reactivity with an α-Hu uRBP Ab.11 This antibody has also been used in a commercially available sandwich ELISA assaya to detect RBP in the urine of healthy (H) cats, untreated hyperthyroid (HT) cats, and cats with chronic kidney disease (CKD).12 Previously, we demonstrated that H cats did not have RBP concentrations above the ELISA limit of quantification (LOQ), whereas the majority of HT cats and cats with CKD had elevated uRBP concentrations, with large variations among individual cats.12 These differences indicate that uRBP might be suitable as a marker for studying the localization of tubular lesions that occur because of kidney damage in cats with CKD or HT cats. Nevertheless, it remains unclear why RBP is present in urine of untreated HT cats.
Plasma concentrations of RBP and transthyretin are decreased in humans with hyperthyroidism, although they remain unchanged in hypothyroid patients.13–15 The decreases in RBP and transthyretin concentrations can be explained either by an increased plasma turnover of RBP combined with unchanged RBP synthesis16 or by lower hepatic synthesis of RBP because of decreased serum zinc values.15 Urinary RBP/creatinine (uRBP/c) ratios decline in HT humans that become euthyroid, but the absolute concentrations vary widely among individuals and the median values of both HT and euthyroid patients are within reference ranges for euthyroid subjects.17
It is possible that HT cats have increased clearance of circulating RBP that leads to or contributes to elevated uRBP. It is also possible that elevated uRBP in HT cats is caused by an effect of the HT state on tubular function. Hyperthyroidism affects other aspects of tubular function.18–21 Therefore, we would expect to see a decrease in uRBP after treatment for hyperthyroidism. Consequently, calculating the fractional excretion (FE) of RBP might help to elucidate a possible link between serum RBP and uRBP.22
The α-Hu uRBP Ab reacts with serum, plasma, and uRBP in humans, and it has been applied in ELISA to measure RBP in both serum and urine.23,24 However, in cats, it has only been used to qualitatively detect RBP by Western analysis of plasma11 and sera,10 and for quantitative analysis of RBP in urine.12 No data are available about the influence of treatment for hyperthyroidism on systemic and uRBP concentrations in cats. Therefore, the objectives of the current study were to compare serum and uRBP concentrations between HT and H cats and to evaluate the influence of radioiodine (131I) treatment on serum and uRBP concentrations in HT cats.
Materials and Methods
This study was approved by the Local Ethics Committee of Ghent University. The care and use of all animals complied with local animal welfare laws, guidelines, and policies. Informed consent was obtained from the owners of all HT cats included in this study.
The 10 HT cats included in the study ranged in age from 8 to 16 years (median 13 years) and weighed 2.6–5.0 kg (median 3.5 kg). There were 2 castrated males and 8 spayed females, all domestic shorthair. Cats were included in the study when diagnosed with hyperthyroidism and presented for treatment with 131I at the Faculty of Veterinary Medicine of Ghent University (Belgium). The success of the therapy was evaluated 24 weeks after treatment based on the decrease in serum total thyroxine (TT4) concentration and the amelioration of clinical signs.
Diagnosis of hyperthyroidism was based on clinical signs compatible with hyperthyroidism, including increased serum TT4 concentration (reference range 1.1–3.5 μg/dL) and increased thyroid uptake (ratio thyroid uptake/salivary gland uptake) of pertechnetate (99mTcO4−). Anti-thyroid drugs had to be discontinued for at least 3 weeks before inclusion. To assess the clinical condition, cats underwent routine physical and laboratory examinations (CBC, biochemistry, and measurement of serum TT4) and cystocentesis for urinalysis 1 day before and 4, 12, and 24 weeks after 131I treatment. At these re-evaluations, serum TT4, serum creatinine, serum RBP, uRBP, and urinary creatinine concentrations were measured. Before and after treatment, urine specific gravity (USG) and glomerular filtration rate (GFR) were measured with plasma clearance of exo-iohexol (PexICT), as described earlier.25
Eight H cats ranging in age from 2 to 10 years (median 9.5 years) and weighing from 2.3 to 5.8 kg (median 4.8 kg) were obtained from the population of laboratory animals of the Faculty of Veterinary Medicine of Ghent University. Three were neutered males and 5 were female (3 spayed, 2 intact); all were Domestic Shorthair. Cats underwent routine physical and laboratory examinations (CBC, biochemistry, and measurement of serum TT4) and cystocentesis for urinalysis. Animals were included only if these examinations showed no clinically significant abnormalities. Serum TT4, serum creatinine, serum RBP, uRBP, urinary creatinine concentration, USG, and GFR (PexICT, n = 7) were measured and the results were compared with those of the HT cats.
Procedures and RBP Analysis
Blood was taken by jugular venipuncture and urine by cystocentesis on the same day after the cat had been fasted for at least 10 hours. No chemical restraints were used for sampling. Blood samples were allowed to clot for a maximum of 1 hour. Clotted serum and urine were stored at 4°C for a maximum of 2 hours. After centrifugation (5 minutes at 2,431 ×g for serum, 3 minutes at 447 ×g for urine), samples were aliquoted and stored at −20°C (serum) or −80°C (urine). Serum TT4 was measured by a validated chemiluminescent immunoassay.b Creatinine was measured in serum and urine by a validated spectrophotometric Jaffé method.c Within- and between-run coefficients of variation (CV) are described in Table 1.
Table 1. Correlation coefficients for low- and high-range concentrations of serum creatinine and serum TT4.
CV, coefficient of variation; TT4, total thyroxine.
Western analysis was performed as previously described for feline urine.12 Briefly, after dot blot optimization (data not shown), 20-μL samples of diluted serum (5–10 μg total protein) from H and HT cats were subjected to 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition to colorimetric and western protein standards, 3 μL of a 0.1-μg/μL uRBP standardd was included in the run. After SDS-PAGE, the separated proteins were electroblotted onto a nitrocellulose membrane with a transfer buffer containing 20% methanol. Before immunodetection, Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% milk powder was used to block nonspecific binding sites on the blot. The membrane was then incubated overnight at 4°C with diluted (1 : 100) polyclonal rabbit α-Hu uRBP Ab, the same antibody as used in the RBP ELISA.e After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1 : 1,000) and then washed with TBS-T. Chemiluminescence was measured after addition of peroxide buffer and luminol enhancer.f
A polyclonal rabbit α-Hu uRBP Ab was used in a commercial sandwich ELISAa validated for assessing RBP in feline urine, and previously described in detail.12 Wells were filled with 100 μL of either 1 : 10 diluted urine samples or 1 : 200 diluted serum samples. The absorbance of each well was measured at 450 nm in an ELISA plate reader, by 600 nm as the reference wavelength. Samples that produced an absorbance <10 standard deviation (SD) of negative control samples were considered as below the LOQ (value 0), as described previously by van Hoek et al.12 The recovery of RBP was quantified with a standard curve generated from serial dilutions of a serum sample taken from an HT cat before treatment. This approach has been validated in dogs.26 RBP concentration is expressed as μg/L for serum and as RBP/c (10−2 μg/mg creatinine) ratio for urine. FE of uRBP was calculated as the fraction of the amount of uRBP filtered through the glomeruli and excreted in the urine with the following formula: (uRBP concentration × serum creatinine concentration)/(serum RBP concentration × urinary creatinine concentration).22
The groups of H and HT cats were compared regarding age and body weight (BW) by t-test and regarding sex by Fisher's exact test.
Serum RBP and TT4 concentrations, uRBP, uRBP/c ratio, USG, and GFR were analyzed with a linear mixed modelg with cat as the random effect and treatment and time as categorical fixed effects. The measurements from the HT cats at the different time points were compared with those from H cats at time 0 by Dunnett's multiple comparisons technique. Additionally, a multivariate analysis was performed by the same model and introducing BW, age, and sex as covariates in order to adjust for imbalances in these covariates between HT and H cats. Tukey's multiple comparisons technique was used to perform pair-wise comparisons of the measurements taken for HT cats at the different time points. All tests were done at a global significance level of 5% and adjusted P-values (adjusted for multiple comparisons) are reported. Pearson's correlation coefficients were obtained for different pairs of variables. Results are expressed as mean ± SD unless stated otherwise.
Western blot analysis of serum samples from H and HT cats showed a distinct band at the position corresponding to that of the RBP standard. The qualitative Western blot analysis was followed by a quantitative ELISA. First, recovery of feline RBP was demonstrated by the parallelism between the curve of serially diluted HT cat serum before 131I treatment and that of human RBP standards. The results indicated that the antigen measured in feline serum was RBP and confirmed the antigen's cross-reactivity with the primary α-Hu antibody, as previously shown for cat urine (Fig 1).12 Serum RBP and TT4 concentrations, uRBP concentrations, uRBP/c ratio, USG, and GFR of H cats and HT cats before and after treatment are presented in Table 2. Serum TT4 concentration differed between H and HT cats before treatment (P < .001) but not 4 weeks (P= .999), 12 weeks (P= 1.000), or 24 weeks (P= .998) after treatment. Serum RBP did not differ significantly between H and HT cats before (P= .984) or 4 weeks (P= .625), 12 weeks (P= .857), or 24 weeks (P= .999) after treatment. Urinary RBP differed significantly between H and HT cats before treatment (P= .001), but not at 4 weeks (P= .491), 12 weeks (P= .385), or 24 weeks (P= .241) after treatment. There was a significant difference between H and HT cats in uRBP/c before treatment (P= .003), but not at 4 weeks (P= .945), 12 weeks (P= .796), or 24 weeks (P= .302) after treatment. FE was higher in HT cats than in H cats at all time points; however, FE did not differ significantly between H cats and HT cats before (P= .131) or 4 weeks (P= .997), 12 weeks (P= .895), or 24 weeks (P= .092) after treatment. USG did not differ between H and HT cats before (P= .834), or 4 (P= .580), 12 (P= .288), or 24 (P= .939) weeks after treatment. GFR differed between H and HT cats at time point 0 (P= .002), but not at 4 (P= .467), 12 (P= .310), or 24 (P= .340) weeks after treatment.
Table 2. Concentrations (mean ± SD) of serum TT4, serum RBP, serum creatinine, urinary RBP, urinary RBP/c ratio, USG, and GFR in H cats (n = 8) and HT cats (n = 10) before (0) and 4, 12, and 24 weeks after 131I treatment.
There was no significant difference between the H and HT cats regarding age (P= .715), BW (P= .06), or sex (P= 1). These results were reanalyzed by multivariate analysis with age, BW, and sex as fixed effects. The estimated differences between the H and HT cats and related P-values were similar in the univariate and multivariate models; therefore, the results are not presented separately.
There was a significant decrease in serum TT4 concentration in HT cats at all time points after 131I treatment relative to before 131I treatment (P < .001). No statistically significant differences in serum TT4 concentration were observed between 4 and 12 weeks (P= 1), 4 and 24 weeks (P= .986), or 12 and 24 weeks after treatment (P= .996). Serum RBP concentration did not change significantly after 131I treatment (P= .799). Compared with pretreatment values, there was a significant decrease in absolute uRBP concentration 4 weeks (P= .004), 12 weeks (P= .006), and 24 weeks (P= .016) after 131I treatment. No statistically significant differences were observed between 4 and 12 weeks (P= .995), 4 and 24 weeks (P= .916), or 12 and 24 weeks after treatment (P= .977). RBP was present in the urine of 50% of the HT cats until 24 weeks after treatment, although the uRBP/c ratios of 4 of these 5 cats were lower than pretreatment ratios. Relative to pretreatment values, there was a significant decrease in uRBP/c ratio at 4 weeks (P= .004) and 12 weeks (P= .001) but not 24 weeks (P= .084) after 131I treatment. No statistically significant differences were observed between 4 and 12 weeks (P= .986), 4 and 24 weeks (P= .580), or 12 and 24 weeks after treatment (P= .781). No significant change in FE were detected at 4, 12, or 24 weeks after 131I treatment (P= .061). There was no significant difference in USG at 4, 12, or 24 weeks after treatment (P= .426). Compared to pretreatment values, GFR decreased significantly in HT cats at 4 (P < .001), 12 (P < .001), and 24 (P < .001) weeks after treatment. No statistically significant differences in GFR were observed between 4 and 12 weeks (P= .158), 4 and 24 weeks (P= .203), or 12 and 24 weeks after treatment (P= .999).
Pearson's correlation coefficients were calculated for all parameters at all time points; the results are presented in Table 3. The correlation between serum TT4 concentration and uRBP/c in HT cats is shown in Figure 2.
Table 3. Pearson's correlation coefficients (r) and corresponding P-values for differences relative to time point 0 for the comparison of serum RBP to serum TT4 and uRBP/c and the comparison of serum TT4 to uRBP/c.
All Time Points
+ 4 Weeks
+ 12 Weeks
+ 24 Weeks
Correlation coefficients were calculated for all time points together, as well as before, 4, 12, and 24 weeks after treatment.
r is significantly different relative to time point 0.
Two HT cats developed CKD (International Renal Interest Society stage II, serum creatinine 1.6–2.8 mg/dL), low USG (1.012 and 1.015, respectively), and clinical symptoms of CKD. Serum creatinine increased from 0.94 to 2.68 mg/dL and from 0.58 to 1.80 mg/dL, respectively. Urinary RBP was present in one of these cats at 24 weeks after treatment. Both cats were also diagnosed with hypothyroidism, defined as serum TT4 concentration below the reference range and no response to rhTSH stimulation.27
Results of this study suggest that untreated HT cats had a significantly higher uRBP concentration than H cats, and that uRBP/c correlated with serum TT4 concentration. Urinary RBP decreased after 131I treatment. However, uRBP/c values were not significantly correlated to serum RBP concentrations before or after treatment. Moreover, serum RBP concentrations were highly variable among HT cats and did not differ significantly from concentrations in H cats. The observation that uRBP in HT cats was not significantly correlated to the systemic RBP concentration suggests that uRBP likely reflects dysfunction at the local tubular level. Reversibility of this renal dysfunction is indicated by the decrease in uRBP/c ratios after 131I treatment; this alteration in tubular function was not unexpected. Indeed, thyroid hormones stimulate active carrier-mediated tubular processes by increased gene expression, synthesis, and activity of carrier proteins like Na+-K+-ATPase and the Na+/H+ exchanger in brush border membrane vesicles.18–20 In addition, the metabolic level and reabsorbtive capacity of tubular cells is increased in hyperthyroidism.21
In HT cats, several factors such as decreased muscle mass, increased renal blood flow, and decreased USG could influence urinary creatinine concentrations. Thus, these factors could have indirectly contributed to the observed differences in the uRBP/c ratios between H and HT cats. However, the absolute uRBP concentration in HT cats decreased significantly after treatment. Moreover, there was no difference between the pre- and posttreatment USG in the HT cats, which supports our hypothesis that a hyperthyroidism-associated reversible tubular defect was responsible for the differences in uRBP before and after 131I therapy.
The FE of RBP was higher in HT cats before and after treatment as compared to H cats, although the differences were not significant. Renal dysfunction, tubular impairment in particular, can increase the FE. Nonetheless, the FE values in animals with impaired renal function are often normal.28,29 To our knowledge, this is the first study to investigate the influence of treatment on serum and uRBP concentrations in HT patients, either humans or animals.
The decrease in feline uRBP/c ratios after radioiodine treatment for hyperthyroidism corroborates results reported for humans.17 The uRBP/c ratio is higher in humans with hyperthyroidism than in subjects with normal thyroid function; however, the value of the ratio varies widely and does not differ significantly from values for control subjects. It is possible that these HT humans had a less advanced stage of hyperthyroidism with less substantial structural tubular damage as compared to the HT cats in the current study. Nevertheless, the mechanism responsible for this change, whether in humans or animals, remains unclear.
In addition to local tubular dysfunction, several systemic factors could contribute to the observed changes in the uRBP/c ratios in HT cats. Any change in the affinity between transthyretin and holo-RBP would lead to an increase in unbound RBP, which is susceptible to glomerular filtration. Malnutrition decreases transthyretin concentrations, thereby increasing the glomerular filtration of RBP.30,31 Undernutrition leading to a low body condition score is common in HT cats. Therefore, although no changes in total serum RBP were observed in our study, we cannot exclude that in untreated HT cats the unbound RBP fraction could be increased, resulting in RBP excretion.8,32 To our knowledge, such a phenomenon has not yet been described.
Several independent studies have described the development of posttreatment renal azotemia in HT cats.33–35 Therefore, it would be of interest to detect pre-existing yet masked renal dysfunction in HT cats. The large individual variation, albeit smaller than in humans,17 in the uRBP/c ratios of HT cats before and after treatment suggests that this ratio could serve as a predictive marker for CKD post 131I treatment. Interestingly, 20% of the HT cats in the current study developed CKD after treatment. Despite the fact that 2 cats had an increased uRBP/c before treatment, the elevation of the uRBP/c persisted until 24 weeks after treatment in only 1 of these 2 cats. A recent study evaluated the usefulness of urinary markers, including uRBP, in a larger number of cats and during prolonged follow-up; the objective was to evaluate the predictive value of different standard and candidate renal markers, including uRBP/c, for the development of posttreatment renal azotemia. Significant changes in kidney function occurred within 4 weeks posttreatment and none thereafter. Pretreatment measurement of GFR, USG, and serum TT4 can have predictive value regarding the development of posttreatment renal azotemia.36 In our study, there was no change in serum TT4 or uRBP/c after more than 4 weeks after treatment. This time point has been shown to be accurate for evaluation of kidney function in HT cats after treatment, because significant changes occurred within 1 month after treatment.36
Indeed, although the systemic transport system of the holo-RBP-transthyretin complex is similar in all mammals, including Felidae, there are important immunological differences among mammalian orders. According to 1 comparative immunology study, feline serum RBP failed to cross-react with a rabbit α-Hu serum RBP Ab in a radioimmunoassay.37 This observation contrasts that from a more recent study in which partial cross-reactivity was seen between a rabbit α-Hu Ab and serum RBP in cats in a radial immunodiffusion assay.10 Feline serum RBP cross-reacted with both α-Hu unbound uRBP Ab as well as to α-Hu serum transthyretin-RBP Ab. It is possible that the α-Hu Ab not only binds to RBP-4, as in humans, but also to other forms of RBP present in serum of cats. With Western blot analysis we excluded the potentially nonspecific binding of the α-Hu Ab to other proteins in feline serum, which is indicative of its specific binding to circulating feline RBP. Different forms of circulating RBP cannot be distinguished by Western blotting.38
In addition to the differences observed among mammals, assay-related factors can also influence the crossreactivity between feline RBP and α-Hu uRBP Abs. Affinity of the α-Hu uRBP Ab for RBP in serum is lower than for uRBP because serum RBP is complexed to transthyretin, while urine RBP is unbound.39 Transthyretin could influence RBP-Ab binding under nondenaturing conditions, as in ELISA.40 Moreover, uRBP is modified by proteolysis at its carboxyl terminus which inhibits binding to transthyretin, and differences in amino acid composition influence the binding of Abs to the antigen.40 Additional influences on serum RBP assessment include the method of serum or plasma collection and the fasting versus fed state of the patient.40 In the current study, serum tubes without clot-activator were used, as recommended, and cats were fasted overnight before blood collection. Burri et al10 found little or no influence of overnight fasting on RBP concentration in humans and rats.
This longitudinal study is the first to report the quantitative assessment of RBP in the serum and urine of H and HT cats before and after treatment for hyperthyroidism. A limitation of our study is the relatively small number of cats evaluated. Thus, when differences were not significant and the null hypothesis could not be rejected, the possibility that a significant difference does exist cannot be excluded. However, significant differences (at the 95% confidence level) in uRBP in HT cats before and after treatment and relative to H cats were found; therefore, we can conclude that these differences are real, even with the small sample size used in this study.
We conclude that the presence of uRBP in HT patients is a potential marker of tubular dysfunction that is correlated to thyroid status, although uRBP appears to be independent of circulating RBP concentration. We interpret from our data that the observed tubular dysfunction in HT cats is mainly reversible by treatment with 131I. Additional data are required to support this hypothesis and to determine the usefulness of RBP as a marker predictive of posttreatment renal azotemia.
aImmundiagnostik AG, Bensheim, Germany
bImmulite 2000 Canine total T4 assay, Diagnostic Products Corporation, Los Angeles, CA
cModular, Roche Diagnostics, Mannheim, Germany
dSigma-Aldrich, Bornem, Belgium
eA0040, DakoCytomation, Glostrup, Denmark
fSupersignal West Dura substrate, Pierce Science, Aalst, Belgium