Utility of Serum Cystatin C as a Clinical Measure of Renal Function in Dogs
A human kit for cystatin C determination was evaluated for use with canine sera. A reference range was also established. The association between cystatin C and glomerular filtration rate (GFR) was evaluated in 60 dogs with various diseases, by using exogenous creatinine plasma clearance (ECPC) as a measure of GFR. The correlation between cystatin C and ECPC (correlation coefficient [r] = −0.630; P<0.001) was stronger than the correlation between serum creatinine and ECPC (r = −0.572; P<0.001). Nonrenal diseases (e.g., neoplasia, infection) did not influence serum cystatin C concentration. Test sensitivity was significantly better (P<0.001) for cystatin C (76%) than for creatinine (65%). Specificities for the two tests were 87% and 91%, respectively.
Introduction
Glomerular filtration rate (GFR) is widely accepted as the most accurate measure of renal excretory function. Renal clearance is defined as the volume of plasma that has been cleared of a particular substance per unit of time, with GFR estimated by renal clearance of an appropriate filtration marker. The changing concentration of this marker can be measured in plasma and urine or in plasma alone.1 However, available methods for estimating GFR are impractical for widespread clinical use. Therefore, serum creatinine concentration is most often used to assess renal excretory function. This test is reasonably specific but somewhat insensitive for detecting reduced GFR.2,3 Creatinine originates from the skeletal muscle and, to a lesser extent, food, so blood levels are influenced by muscle mass and (to a lesser extent) food intake.2
Cystatin C is a protease inhibitor that belongs to the family of cystatines. Together with the stefines and kinogenes, the cystatines form the super-family of cysteine proteinase inhibitors.4 The cystatin C molecule consists of a single polypeptide chain having 120 amino acids and a molecular weight of 13.359 kilodalton (kDa). The molecule is not glycosylated, and it has an isoelectric point of 9.3.5 Cystatin C is produced by all nucleated cells within the body and is released during phagocytosis and inflammation.
The major function of cystatin C is to control inflammation by inhibiting lysosomal proteases.6 Cystatin C helps to regulate intra- and extracellular protein/peptide catabolism, as well as the penetration of cancer cells into tissues.7 Recent studies have also demonstrated antimicrobial properties associated with this protein.4,8–10 The rate of cystatin C synthesis is constant (housekeeping type),11,12 independent of age and gender, 13 and unaltered by inflammatory processes or neoplasia.11 High concentrations can be found in serum, seminal fluid, cerebrospinal fluid (CSF), and synovial fluid.14
Cystatin C fulfills many criteria of an ideal GFR marker, because it is filtered by the glomeruli without any tubular secretion. The molecule passes through the glomeruli easily because of its low molecular weight and positive charge at physiological pH. Cystatin C is catabolized completely within the proximal tubular cells, so urine concentration is typically low13,15 and unaltered by diseases of the proximal tubules.16 Cystatin C is eliminated exclusively by the kidneys17 and is superior to serum creatinine as a marker of GFR in people.13,18–21 However, few published studies explore the diagnostic utility of cystatin C in dogs.22,23
The main goals of the present study were to validate a human cystatin C test kit in dogs and to establish a canine reference range. A third goal was to compare serum cystatin C concentrations with exogenous creatinine plasma clearance (ECPC) to assess its use as a measure of renal function.
Materials and Methods
Validation of the Test Kit
The test kit was validated using a particle-enhanced turbidimetric (PET) assaya designed to quantify cystatin C levels in human serum or plasma. In this technique, cystatin C binds to rabbit antihuman cystatin C antibody that has been coupled to polystyrene particles. The resulting change in turbidity is proportional to the cystatin C concentration in the sample. The assay was set up on an automatic analyzerb according to the manufacturer’s instructions.
Two serum pools were created to evaluate measures of assay quality. One pool consisted of sera from eight dogs with increased serum creatinine concentrations, and the other consisted of sera from eight dogs with normal values. Interassay variability was assessed by freezing 11 serum samples (−80°C) from each of the two serum pools and then testing one daily for 10 consecutive days, with the 11th sample thawed and tested after 365 days. Dilutional parallelism was determined by serial dilution with physiological saline, so as to produce concentrations that were 75%, 50%, and 25% of the original. Determining cystatin C recovery was not possible, because a purified source of canine cystatin C was not available, and the authors’ research facilities were not equipped to purify it.
To evaluate intraindividual variation, blood samples were obtained at 0, 360, and 720 minutes from 27 dogs with various degrees of kidney function. All dogs had been fasted for 12 hours.
Establishment of Reference Ranges
Blood samples were taken from 99 dogs presented for annual checkup examinations. All dogs had been fasted for 12 hours and had normal physical examinations, complete blood counts, biochemical profiles, and urinalyses (i.e., stick, specific gravity, sediment). Blood and urine were collected by jugular vein puncture and cystocenteses, respectively. Serum samples for cystatin C measurement were frozen (−80°C) until analyzed.
Owners gave informed consent before any blood and urine samples were obtained from dogs, in accordance with published laws and university policy.
Comparison of Cystatin C and ECPC in Dogs
Ideally, GFR should be assessed with a urinary inulin clearance, which is considered the gold standard. However, urinary clearance testing was considered impractical in a hospital facility, so GFR was assessed by ECPC, which is a crude but accepted substitute.24
Exogenous creatinine plasma clearance was performed in 60 dogs that had been diagnosed with various renal and nonrenal diseases. Of these dogs, 27 were suspected of having chronic kidney disease, with the remaining 33 being assessed for renal function before treatment with potentially nephrotoxic substances. Final diagnoses were based on histopathology/cytology (n=24), serology (n=11), ECPC (n=17), and other methods (e.g., endocrine testing, abdominal ultrasonography, and echocardiography). Dogs had been fasted for 12 hours before examination. All were clinically well hydrated.
A standardized procedure was used to assess all 60 dogs. First, serum creatinine concentration was determined, followed by intravenous injection with a 5% creatinine solutionc dosed at 60 to 125 mg/kg. The exact dosage depended on body mass, with lower concentrations applied to larger dogs. Twelve to 15 blood samples were obtained over the next 10 hours, so that serum creatinine concentrations could be measured enzymatically.b The upper limit of the in-house reference range for serum creatinine was 106 μmol/L (1.2 mg/dL). The area under the curve (AUC) was calculated with a trapezoidal approach (i.e., noncompartmental model).1
The ECPC was calculated as 100 multiplied by the amount of injected creatinine divided by the AUC (100 × injected creatinine/AUC).24–26 An ECPC ≥3 mL/kg per minute was considered normal. Values of 2.00 to 2.99 mL/kg per minute were considered to be slightly reduced, and values ≤1.99 mL/kg per minute were considered to be markedly reduced. These values were compared with the reference range for cystatin C that was already established (see above).
Statistics
Statistical analyses were performed using version 13.0 of SPSS.d Descriptive statistics were computed for the cystatin C test kit validation and reference range calculations. Kolmogorov-Smirnov analysis was used to determine if the age and gender variables used in the reference range calculations were normally distributed and if the reference range itself was normally distributed. Pearson’s correlation statistics were used to assess the association between cystatin C and age, weight, or gender. Correlational analysis was also used to assess the relationships between ECPC and serum creatinine, between ECPC and cystatin C, and between serum cystatin C and presence of nonrenal disease.
Receiver operating characteristics (ROC) analysis was used to compare sensitivity, specificity, and positive/ negative predictive values for both serum cystatin C and serum creatinine. Sensitivity is defined as true positives divided by the sum of true positives and false negatives, and specificity is defined as true negatives divided by the sum of false positives and true negatives. Positive predictive value is defined as true positives divided by the sum of true positives and false positives, and negative predictive value is defined as true negatives divided by the sum of true negatives and false negatives. Statistical significance was defined as P<0.05.
Results
Validation of the Test Kit
The intraassay coefficients of variation were 1.76% for the serum pool consisting of the eight dogs with high creatinine values, and 3.85% for the serum pool consisting of the eight dogs with normal values. The interassay coefficients of variation for these two groups were 2.95% and 3.64%, respectively [see Table]. Values for the two serum pools were 8% and 11% higher (respectively) after 365 days. An almost linear decrease in cystatin C concentration was observed after serial dilution (75%, 50%, 25%). The correlation coefficient (r) was 0.992 (P<0.01) for those with high creatinine, and r was 0.989 (P<0.05) for those with normal creatinine [Figures 1A, 1B].
Intraindividual Variations
Serum cystatin C concentrations remained stable over time in the 27 dogs sampled at 0, 360, and 720 minutes (r=0.964, 0.954, respectively; P<0.001).
Establishment of Reference Values
The 99 reference dogs ranged in age from 3 months to 13 years (median 4 years). Twenty-five dogs were intact females, 27 were spayed females, 26 were intact males, and 21 were neutered males. Body weight ranged from 5 to 42 kg (median 25 kg). Kolmogorov-Smirnov analysis suggested that age and weight were normally distributed. Breeds represented by more than one case in the reference group were German shepherd dog (n=18); golden retriever (n=8); Labrador retriever (n=6); dachshund (n=4); Magyar vizsla, border collie, Belgian Tervuren, Dalmatian, Siberian husky (n=3 each); and Bernese mountain dog, Irish wolfhound, Schapendoes, boxer, and rottweiler (n=2 each). The remaining 38 cases consisted of other breeds (n=12) or mixed breeds (n=26).
Serum cystatin C concentrations ranged from 0.49 to 1.81 mg/L (mean 1.144, standard deviation [SD] ± 0.231). The 0.025 and 0.975 quantiles (determined as mean ± 2 SD) were 0.682 mg/L and 1.606 mg/L, respectively, suggesting a reference range of 0.68 to 1.6 mg/L. Kolmogorov- Smirnov analysis suggested that cystatin C values were normally distributed.
Serum creatinine concentrations for the 99 dogs ranged from 56 to 103 μmol/L (0.63 to 1.17 mg/dL), with a mean ± SD of 81.90±13.29 μmol/L. A serum creatinine reference range for these 99 dogs would be 55.31 to 108.5 μmol/L (0.63 to 1.23 mg/dL), based on 0.025 and 0.975 quantiles. This almost matched the established in-house reference range, so the upper limit for serum creatinine was left as the in-house value of 106 μmol/L (1.2 mg/dL).
Influence of Age, Weight, and Gender
No significant correlation was found between serum cystatin C and age (r=0.022; P>0.05) or between cystatin C and weight (r=0.036; P>0.05). Serum cystatin C concentrations were similar in the 52 female (mean ± SD 1.13±0.24 mg/L) and 47 male (mean ± SD 1.15±0.21 mg/L) dogs [Figure 2]. The Mann-Whitney U test and Wilcoxon’s test confirmed that the cystatin C distributions were similar between males and females.
Comparison of Cystatin C and ECPC in Dogs
The ages for these 60 dogs ranged from 0.5 to 15 years (median 5 years), and the weights ranged from 7 to 51.8 kg (median 27.2 kg). Twenty-five females (16 spayed) and 35 males (eight neutered) were represented. Breeds that were represented by more than one case were Bernese mountain dog (n=6), boxer (n=4), beagle (n=3), German shepherd dog (n=3), and West Highland white terrier (n=2). The remainder consisted of other purebred (n=15) or mixed-breed (n=27) dogs. These dogs were ultimately diagnosed with neoplasia (n=7), chronic kidney disease (n=24), neoplasia and concurrent chronic kidney disease (n=15), infection (n=10), or four other diseases (two endocrinopathies, one psychogenic drinker, and one mitral insufficiency).
Serum creatinine values for these 60 dogs ranged from 42 to 590 μmol/L (0.46 to 6.67 mg/dL), with a median of 91 μmol/L. Forty-three percent of these values were above the reference range (i.e., >106 μmol/L [>1.2 mg/dL]). Cystatin C values ranged from 0.79 to 5.97 mg/L (median 1.65 mg/L), with 52% having elevated concentrations (>1.6 mg/L). Results of ECPC ranged from 0.3 to 5.0 mL/kg per minute (median 2.6 mL/kg per minute), with 38% (23 of 60) dogs having normal (≥3 mL/kg per minute) values, and 62% (37 of 60) having abnormal values. The abnormal ECPC values consisted of slightly decreased ECPC (i.e., 2.00 to 2.99 mL/kg per minute) in 22 dogs and markedly decreased ECPC (≤1.99 mL/kg per minute) in 15 dogs.
Correlation Among ECPC, Creatinine, and Cystatin C
A significant inverse correlation was seen between ECPC and serum creatinine in these 60 dogs (r= −0.572; P<0.001), which appeared less than linear [Figure 3A]. Also, a significant and less than linear inverse correlation (r= −0.630; P<0.001) was seen between ECPC and serum cystatin C, but the relationship appeared more linear than the one between ECPC and creatinine [Figure 3B]. A strong and significant positive correlation (r=0.886; P<0.001) was seen between serum creatinine and serum cystatin C, which approached linearity [Figure 4].
Correlation Between Creatinine or Cystatin C and the Degree of Renal Function
Dogs were assigned to three groups according to the results of the ECPC. Group 1 had normal ECPC (≥3 mL/kg per minute; n=23); group 2 had slightly reduced ECPC (2.00 to 2.99 mL/kg per minute; n=22); and group 3 had markedly reduced ECPC (≤1.99 mL/kg per minute; n=15). Serum creatinine and cystatin C concentrations were compared within these groups [Figures 5A, 5B]. The median serum concentration of cystatin C in dogs with slightly reduced ECPC (Group 2) was outside the reference range, while the median serum creatinine concentration was within the reference range. All dogs with markedly reduced ECPC (Group 3) had elevated serum cystatin C concentrations, but some had normal serum creatinine values.
Comparison of Cystatin C Among Final Diagnoses
Dogs were assigned to five groups according to their final diagnosis. Cystatin C concentrations in the different groups were compared to explore the influence of nonrenal diseases on cystatin C. Almost all dogs with nonrenal diseases and normal GFR had cystatin C values within the reference range [Figure 6], and all such dogs had normal creatinine values.
Sensitivity, Specificity, ROC Analysis, Positive and Negative Predictive Values
The sensitivity for detecting decreased ECPC (<3.0 mL/kg per minute) was 76% for cystatin C, but sensitivity was only 65% for serum creatinine (P<0.001). Specificities were similar between serum cystatin C (87%) and serum creatinine (91%). The ROC analysis suggests that cystatin C is significantly better (P<0.001) than serum creatinine at identifying reduced ECPC [Figure 7]. The positive and negative predictive values for detecting decreased ECPC were 90% and 69% (respectively) for serum cystatin C, and they were 92% and 62% (respectively) for serum creatinine.
Discussion
Identification of renal disease is important in canine medicine, but early diagnosis remains difficult in some cases. Serum creatinine concentration rises as GFR falls, but azotemia is not usually clinically apparent in dogs until GFR decreases by ≥75%. Therefore, creatinine screening tests may not detect mildly decreased renal function. Mild kidney disease in dogs can be reliably diagnosed only by performing time-consuming clearance procedures, such as ECPC or iohexol plasma clearance. In contrast, human studies have focused on endogenous, low-molecular-weight proteins as a way to detect early renal dysfunction.18 Multiple studies of GFR reduction in people have shown that serum cystatin C concentration has better sensitivity and negative predictive value than serum creatinine.16,27–30
The authors’ study suggests that the human PET assaya is valid for measuring cystatin C in dogs. This test is rapid, precise, and inexpensive. It measures cystatin C in a linear and proportional manner, which is consistent with results of other veterinary studies.22,23 Unfortunately, the authors were unable to determine percent recovery at their facility. This is a limitation of this study, in that the authors were not able to definitively prove that the PET assay was measuring cystatin C.
The exact genetic structure of canine cystatin C is unknown, but a high degree of homology appears to exist between people, cattle, mice, and rats. One genetic sequence (i.e., the so-called μ-trace) has a high degree of homology between people and dogs.31–35 The authors hypothesized that the PET assay might reliably detect canine cystatin C, given this homology between people and dogs and the high degree of correlation between the PET assay and renal function.
This study demonstrated that serum cystatin C is stable over time and can be reliably measured after being frozen at −80°C for 1 year. The same has been shown in people.36 High concentrations of proteinase inhibitors (e.g., α2- macroglobulin, α1-protease inhibitor, and kinogenes) and natural preservatives, such as transferrin, add stability to the blood.18
The reference range for cystatin C in this study was 0.68 to 1.6 mg/L, which is slightly above the range observed in other veterinary and human studies.22,23,37–39 This slight difference may have been caused by slight variations in the assay principle or reagents or by differences in instrumentation.
Ideally, a GFR measurement should have been performed in the population used to establish the cystatin C reference range, in order to assure that all test dogs truly had normal renal excretory function. However, the 99 dogs used to establish the reference range in this study had no medical complaints and were considered healthy based on physical and laboratory examinations. Further workup (including GFR) on healthy dogs would have also required a petition to the government for an animal experimentation permit. For these reasons, the authors opted to forgo simultaneous assessment of GFR. Standard convention was followed, and the reference range was based on the 0.025 and 0.975 quantiles, which excluded extreme values and decreased the likelihood of including dogs with early, undetected, chronic kidney disease.
Age, body weight, and gender did not influence cystatin C concentration, suggesting no need to calculate ranges dependent on these variables. These findings are similar to those of human studies,40–42 but contrary to those of one veterinary study.39 However, no GFR measurements were performed in that veterinary study,39 suggesting that the possible inclusion of early kidney disease could have impacted the results.
The authors observed low intraindividual cystatin C variation over time, which is an advantage for this test. Low intraindividual variation simplifies the collection of samples, which do not need to be drawn at any specific time of day.
Studies in dogs suggest that GFR increases significantly after protein ingestion.43 All dogs in this study were fasted, so the authors could not investigate the effects of food on cystatin C levels. In a human study, cystatin C remained relatively stable despite food consumption.41 However, a study in dogs demonstrated a significant decrease in cystatin C levels after feeding.39 This discrepancy might be explained by the different protein concentrations in human meals compared with those in dog food. More data are needed that examine the impact of feeding on serum cystatin C concentrations in dogs.
None of the dogs in this study had acute kidney disease. In people with acute kidney disease, serum cystatin C levels rise earlier than serum creatinine levels and correlate better with GFR.44–46 No published veterinary studies have analyzed serum cystatin C in the presence of acute kidney disease, but volume depletion might mimic this condition. Almy et al.23 showed that dogs with volume depletion had cystatin C concentrations within the reference range, despite a mildly reduced GFR. However, it is not known if volume depletion adequately reflects acute kidney disease. More studies are needed to investigate the influence of acute kidney disease, volume depletion, and postrenal azotemia on serum cystatin C concentration.
In this study, serum cystatin C correlated better with ECPC (r= −0.630; P<0.001) than did serum creatinine (r= −0.572; P<0.001), suggesting that cystatin C is probably a more sensitive test of reduced GFR. In other words, in dogs with early renal impairment, cystatin C concentrations will probably increase before serum creatinine concentrations. This is borne out in this study by the 76% sensitivity for detecting reduced GFR, which is 11% higher than that of serum creatinine. Although a sensitivity of 80% or 90% would be more desirable, currently no better test is available to diagnose early renal excretory dysfunction from a single canine blood sample.
Cystatin C and creatinine have similar positive predictive values. However, the higher negative predictive value for serum cystatin C (69%) compared with that of serum creatinine (62%) will result in fewer false negatives, suggesting a better screening test for early canine kidney disease.
The slightly better specificity of serum creatinine (91%) over serum cystatin C (87%) makes the authors reluctant to recommend complete replacement of serum creatinine testing at this time. Using both tests simultaneously should enhance diagnostic accuracy for kidney disease. The similar specificities for both parameters in this study suggest the need for additional studies that confirm the superior specificity of serum creatinine.
In this study, serum cystatin C was not influenced by neoplasia or infection. Although cystatin C is involved in inflammation and spread of cancer cells, studies in people have not found that these conditions alter serum concentrations; 11,12 results of this current study agree. However, the authors examined only a limited number of dogs with neoplastic or infectious disease, suggesting that more data are needed to better assess the impact of these diseases on serum cystatin C concentration.
Conclusion
Endogenous, low-molecular-weight proteins could serve as a practical way to rapidly detect early kidney disease. Canine cystatin C was reliably measured with the human PET assay,a independent of age, body weight, gender, or the presence of neoplastic/infectious disease. Serum cystatin C correlated slightly better with ECPC than did serum creatinine. Cystatin C had a higher sensitivity and negative predictive value than did serum creatinine, making it a better screening test for early canine kidney disease. The slightly better specificity of serum creatinine suggests that both cystatin C and creatinine should be measured simultaneously in order to enhance diagnostic accuracy for detecting (or excluding) canine renal excretory dysfunction.
Particle-enhanced turbidimetric (PET) assay; Dako, Glostrup, Denmark
Hitachi 911 automatic analyzer; Roche, Mannheim, Germany
Creatinine anhydrous 5% solution; Sigma-Aldrich, Hamburg, Germany
SPSS 13.0; SPSS Inc., Chicago, IL 60606
Acknowledgments
The authors thank Drs. Michael D. Willard and George E. Less for their contributions to the manuscript.
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
![Figure 2—. Cystatin C concentrations (mean ± standard deviation [SD]) of 99 clinically normal dogs (52 female and 47 male) used to establish a reference range.](/view/journals/aaha/44/3/134_fig2.jpeg)
![Figure 2—. Cystatin C concentrations (mean ± standard deviation [SD]) of 99 clinically normal dogs (52 female and 47 male) used to establish a reference range.](/view/journals/aaha/44/3/full-134_fig2.jpeg)
![Figure 2—. Cystatin C concentrations (mean ± standard deviation [SD]) of 99 clinically normal dogs (52 female and 47 male) used to establish a reference range.](/view/journals/aaha/44/3/inline-134_fig2.jpeg)
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Citation: Journal of the American Animal Hospital Association 44, 3; 10.5326/0440131
Correlation between the observed and calculated cystatin C concentrations of different dilutions of pooled canine sera (1A=pool with higher concentrations, n=8; 1B=pool with normal concentrations, n=8).
Cystatin C concentrations (mean ± standard deviation [SD]) of 99 clinically normal dogs (52 female and 47 male) used to establish a reference range.
Correlation between exogenous creatinine plasma clearance (ECPC) and either serum creatinine concentrations or serum cystatin C concentrations in 60 dogs with various diseases. The area between the vertical black lines indicates mild kidney disease. The dashed lines indicate the upper reference ranges for serum creatinine and serum cystatin C.
Correlation between serum creatinine and serum cystatin C concentrations in 60 dogs with various diseases.
Serum creatinine and serum cystatin C concentrations in 60 dogs with various diseases, according to renal function as defined by exogenous creatinine plasma clearance (ECPC) measurement. The dashed lines indicate the upper reference ranges for creatinine and cystatin C. Normal renal function is defined as ECPC ≥3 mL/kg per minute; slightly impaired renal function is defined as ECPC 2.00 to 2.99 mL/kg per minute; and markedly impaired renal function is defined as ECPC ≤1.99 mL/kg per minute.
Serum cystatin C concentrations in 60 dogs with different diseases. The dashed line indicates the upper reference range for cystatin C; CKD=chronic kidney disease.
Nonparametric receiver operating characteristics (ROC) plots of percent sensitivity and specificity for discriminating between normal and reduced exogenous creatinine plasma clearance (ECPC). The sensitivity of serum cystatin C is significantly (P<0.001) higher than that of serum creatinine in 60 dogs with various diseases.
