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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Objectives: To evaluate the performance of a particle-enhanced turbidimetric assay for measuring canine urinary cystatin C and to investigate if the urinary cystatin C to creatinine ratio is higher in dogs with renal disease than in non-renal disease dogs.

Methods: Urinary cystatin C was measured by particle-enhanced turbidimetric assay using an avian antihuman cystatin C antibody and the performance of this assay was evaluated. Clinical relevance was tested in 46 dogs that were divided into three groups: healthy dogs (n=14), non-renal disease dogs (n=17) and dogs with renal disease (n=15).

Results: The assay was linear (R2=0·99) and precise (mean intra- and inter-assay coefficients of variation were 2·3 and 2·9%, respectively). The recovery was 111·5% and the limit of blank was 0·02 mg/L. Urinary cystatin C and urinary cystatin C to creatinine ratio differed significantly (P<0·001) between the three cohorts of dogs.

Clinical Significance: Measurement of cystatin C by particle-enhanced turbidimetric assay performed with high precision and linearity. This assay can be processed on automated clinical chemistry analysers making it widely available to commercial laboratories. Urinary cystatin C to creatinine ratio can differentiate dogs with renal disease from dogs without renal disease. These preliminary results suggest that urinary cystatin C to creatinine ratio is a promising marker for evaluating renal tubular function.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

In the past number of years, serum cystatin C (SCysC) has been extensively investigated as a potential biomarker of glomerular filtration rate (GFR). A previous study in veterinary medicine suggested that this analyte had a better sensitivity and higher negative predictive value compared to creatinine for documenting decreased GFR (Wehner and others 2008). Almy and others (2002) found a closer correlation between the reciprocals of cystatin C and GFR than that of the reciprocals of creatinine and GFR 2 weeks after reduction of renal mass but, at 10 weeks, creatinine and cystatin C correlated equally well. Unlike creatinine, SCysC is unaffected by muscular mass and gender and is less sensitive to analytical interference (Vinge and others 1999, Braun and others 2002) although a recent paper showed that the biological variance of cystatin C and creatinine in dogs is similar (Pagitz and others 2007).

Cystatin C is a low molecular weight non-glycosylated basic protein that belongs to the super family of cysteine protease inhibitors (Koyner and others 2008). It is encoded by a housekeeping-type gene and is synthesised with a stable production rate by most nucleated cells. In humans, a few extra-renal factors have been proven or suspected to influence the serum concentration of cystatin C, mainly by altering its rate of production. Thyroid function and corticosteroid therapy are recognised to affect the rate of synthesis of the molecule (Bokenkamp and others 2002, Wiesli and others 2003). Furthermore, it has been suggested in humans that circulating cystatin C concentration may be affected by malignancies and that tumours such as melanoma and colorectal cancer are associated with an increased concentration (Kos and others 1998). In small animals, it has been reported that cystatin C production is not influenced by inflammation or neoplasia (Wehner and others 2008). Once produced, cystatin C is released into the circulation. Owing to its low molecular weight and its positive charge, cystatin C is freely filtered by the glomeruli. After glomerular filtration, cystatin C is reabsorbed and catabolised by the proximal tubular cells, with the remaining minimal part being eliminated in the urine, where the concentration is therefore low. In tubular damage, which impairs the reabsorption and degradation of cystatin C, it is hypothesised that the urinary concentration of this analyte would increase considerably. The kinetics of canine cystatin C have not yet been described in the veterinary literature. However, for the purpose of this study it was assumed that the production, metabolism and excretion of cystatin C in dogs and human are similar.

The primary aim of this study was to validate a particle-enhanced immunoturbidimetric assay (PETIA) for measurement of canine urinary cystatin C. A second objective was to evaluate the performance of this marker in urine to discriminate dogs with and without renal disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

This study was conducted in accordance with national (UK) and local ethical guidelines.

Animals

Blood and urine samples in excess of amounts required for routine diagnostic testing and with owner consent for use were obtained for this study. Samples from control and renal disease dogs were sourced from three veterinary centres (two veterinary hospitals and one general practice) or were provided by commercial laboratories. For the purpose of this study, serum creatinine, urea and cystatin C and urinary cystatin C (UCysC), creatinine and total protein were measured and recorded. In addition, UCysC and urinary total protein were normalised for urinary creatinine to compensate for differences in diuresis. The collection of urine was random and untimed. Routine complete blood count (CBC), biochemistry and urinalysis were performed within 24 hours of collection. Both SCysC and UCysC were measured within 24 hours of collection until the stability test was carried out. Once storage stability was demonstrated, the samples were analysed within 1 or 3 months of collection if the samples were stored at –20 or –80°C, respectively.

Similar to a previous study on canine SCysC (Jensen and others 2001), dogs were divided into three groups: healthy dogs (group A), non-renal disease dogs (group B) and renal disease dogs (group C). Dogs were defined as healthy if their history, physical examination, CBC, biochemistry and urinalysis which included specific gravity, dipstick, sediment analysis and urine protein to creatinine ratio were unremarkable. Non-renal disease dogs were patients admitted with a disease unrelated to the urinary tract and without a history (past or recent) or clinical signs referable to kidney disease and that did not have concentrations of urea and creatinine above the upper limit of the reference interval at the time of recruitment. Dogs were included in group C if they were azotaemic and the anamnesis and clinical signs were consistent with intrinsic renal disease (pre-renal and post-renal diseases were excluded). The historical signs related to renal failure included polyuria and polydipsia, vomiting, anorexia, and weight loss. Azotaemia was defined as urea and creatinine concentrations above the upper limit of the reference intervals (reference intervals for urea and creatinine are 3·3 to 8 mmol/L and 36 to 120 μmol/L, respectively). All samples received from external institutions were re-analysed for urea and creatinine and the same inclusion criteria were adopted.

Dogs were excluded from the study if they had previously been diagnosed with hypothyroidism, hyperadrenocorticism, or were receiving glucocorticoid therapy.

PETIA of cystatin C

The validation study was performed in a clinical pathology laboratory under the supervision of an ECVCP diploma holder (JA). The measurement of SCysC and UCysC was obtained by using an automated analyser (Olympus AU400, Beckman Coulter) and a human assay kit based on avian antihuman cystatin C antibody coupled to polystyrene particles (Gentian, Moss, Norway). The analyser programme was slightly modified and an additional calibration point was introduced (point 0). The new calibration curve covered the range between 0·00 and 8·00 mg/L, over seven calibration points. The new point 0 was obtained by using 0·9% sodium chloride (0·00 mg/L). Creatinine concentration was measured by the Jaffe kinetic method on the same analyser.

The performance of the assay was assessed by estimating the precision, linearity, recovery and limit of blank. Potential interference by haemoglobin and the stability of the analyte under different storage conditions were verified.

The intra- and inter-assay precisions were evaluated analysing multiple canine urine samples with different concentrations of cystatin C. For the intra-assay precision, five urine samples with different concentrations of analyte were used (mean values for each urine sample were 0·05, 0·23, 1·38, 3·18, and 7·82 mg/L). The intra-assay reproducibility was obtained by using 10 replicate analyses of each urine sample except for one sample which was analysed nine consecutive times. The inter-assay variability was evaluated by analysing each urine sample in triplicate on independent runs over a period of seven consecutive working days. Four urine samples were used, these having cystatin C concentrations varying from low to high (mean values for each urine sample were 0·04, 0·22, 4·38, and 7·45 mg/L). The precision was expressed as coefficient of variation [CV%=(sd/mean)×100].

Linearity was determined over a range between 0·00 and 5·93 mg/L by analysing a canine urine sample with high concentration of cystatin C serially diluted with a urine sample of low concentration. This method was preferred to dilution with saline (sodium chloride at 0·9%) to avoid any possible alteration of the urine matrix. In the absence of purified canine cystatin C, the accuracy of the method was obtained by adding human calibration solution to a canine urine sample with known concentration of cystatin C and calculating the recovery.

The limit of blank was defined by analysing saline solution in five replicates over a period of five consecutive days in order to include in the result the effect of the intra- and inter-assay variabilities. The mathematical formula: LoB=mean+1·65(sd blank) was applied (Armbruster and Pry 2008).

Interference by haemoglobin was verified by measuring in triplicate samples of canine urine with a known amount of cystatin C to which different concentrations of haemoglobin were added. Haemoglobin was obtained from a sodium citrate sample of canine blood after inducing artificial haemolysis.

The analyte stability was tested on two fresh urine samples which were aliquoted and stored at various temperatures for different times: room temperature for 72 hours, +4°C for 7 days, –20°C for 1 month and –80°C for 3 months. The stability of the analytical solution was expressed as the variation of the measured mean concentration as a function of time and was compared to the analytical variation of the assay (CV%). The criterion of acceptability was considered a variation of concentration over time lower or equal to the coefficient of variation (CV) of the assay.

This assay was also re-validated in a similar manner for the measurement of SCysC because the manufacturer’s PETIA reagent had not previously been validated for dogs. Similarly, the intra- and inter-assay precisions of serum and urinary creatinine and serum urea were evaluated.

Before sample analysis, an internal quality control programme was routinely performed using two control materials (low and high concentrations). Currently and at the time of the study, an external quality control programme (EQA) was also in use for all routine biochemistry tests including serum creatinine and urea but this was not available for urinary creatinine. The accuracy of serum creatinine and urea was calculated from the EQA results, while the bias of the urinary creatinine was obtained from the “target values” of the quality control materials provided by the manufacturer.

Statistical analysis

Arithmetic means, medians, sds, CVs and linear regression were obtained by using Excel 2007 (Microsoft Corporation, Seattle, WA, USA). The three cohorts of dogs were compared by using nonparametric statistical tests as indicated (Kruskal-Wallis test or Mann-Whitney U test). The same statistical tests were used to evaluate the effect of age and gender on the concentration of cystatin C in serum and urine of the control dogs. All statistical analyses were performed with statistic software (IBM Company©, SPSS Statistic 19). P values less than 0·05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Animals

On the basis of the inclusion criteria, 14 dogs were classified as healthy and were included in group A. Group B accounted for 17 dogs which were diagnosed with different diseases varying from malignant neoplasia (squamous cell carcinoma, sarcoma, mammary tumour and chemodectoma) to inflammatory conditions (stick injury, aspergillosis, lameness and foreign body). Fifteen dogs with renal disease were allocated to group C. These cases included 13 dogs with chronic kidney disease and 2 dogs with acute renal failure. Age, gender and breed distributions are presented in Table 1. The distribution of age was significantly different across the three groups (Kruskal-Wallis test; P=0·03) with group A being younger than groups B and C.

Table 1. Breed, gender and age of the dogs included in the three groups (healthy: group A, non-renal disease: group B, and renal disease: group C)
GroupBreedGender (M/F)Age
  1. sd, standard deviation; M, male; F, female

ADachshundM10 months
 CrossbreedM13 years
 Labrador retrieverM8 months
 Maltese terrierF2 years
 CrossbreedM2·5 years
 Yorkshire terrierF2 years
 CrossbreedF1·5 years
 Border collieF1 year
 RottweilerM8 years
 German shepherdM8 years
 Border collieM7 years
 PapillonM3 years
 GreyhoundM3·6 years
 GreyhoundF3·5 years
 Age: mean, median and sd4·05, 2·75 and 3·61
BBoxerF15 years
 Labrador retrieverM5·7 years
 German shepherdM9 years
 Bull terrierF4 months
 Flat coated retrieverUnknown9 years
 English setterF13 years
 CrossbreedM8 years
 English setterF6 years
 English pointerM5 years
 Golden retrieverM4 years
 Shih-tzuMUnknown
 PugM4 years
 CrossbreedF11 years
 Cocker spanielM11 years
 Labrador retrieverM1·2 years
 PugF2 years
 CrossbreedF11 years
 Age: mean, median and sd7·13, 7·0 and 4·30
CBorder collieF2·3 years
 Great DaneM6 years
 West Highland white terrierF13 years
 Labrador retrieverF6·4 years
 Border terrierF4 years
 Jack Russell terrierF9.5 years
 German shepherdF5 years
 RottweilerM7·5 years
 English springer spanielM7·6 years
 Rhodesian ridgebackM11 years
 Bichon friseF10 years
 Staff ordshire bull terrierM5 years
 Labrador retrieverF3·4 years
 Old English sheepdogM3·1 years
 Jack Russell terrierM12 years
 Border collieF2·3 years
 Great DaneM6 years
 Age: mean, median and sd7·09, 6·4, and 3·35

There was no significant difference in the distribution of SCysC, UCysC and UCysC:C between males and females in the groups A and B. However, a significant increase of SCysC was demonstrated with increasing age across the different categories of age divided in quartiles (Kruskal-Wallis test; P=0·015). Age was not a significant factor in UCysC and UCysC:C.

PETIA measurement of cystatin C in urine

The intra-assay analytical imprecision varied from 1·4 to 4·21% (from the lower to the higher UCysC concentration, the CV% was respectively 4·21, 1·40, 1·80, 1·90, and 2·10%) and the inter-assay imprecision ranged from 2·11 to 3·8% (3·8, 2·25, 2·11 and 3·64% from the lower to the higher UCysC concentration). The mean intra- and inter-assay CVs were 2·3 and 2·9%, respectively. The measurement of cystatin C in the range 0·00 to 5·93 mg/L was linear (R2=0·99). The recovery method was 111·5% and the LoB was 0·02 mg/L. All samples with a concentration below the LoB were assigned a value of 0·02 mg/L for further analysis. The interference study proved that the measurement of cystatin C is not altered by haemoglobin up to a concentration of 400 g/L. The stability test showed that cystatin C was stable in canine urine for 72 hours at room temperature, a week at 4°C, 1 month at –20°C and 3 months at –80°C with a variation lower than 1·7%. The intra- and inter-assay mean CVs for measurement of SCysC were 3·5 and 2·4%, respectively. The overall imprecision of serum creatinine was less than 1%, that of urinary creatinine was 1·6% and of urea was 1·4%. Accuracy of serum and urinary creatinine were, respectively, 103 and 97% while the accuracy of urea was 104%.

Clinical study

The range of SCysC values was 0·18 to 1·21 mg/L in group A, 0·24 to 1·42 mg/L in group B and 0·73 to 9·06 mg/L in group C. The urinary cystatin C ranges were <0.02-0.26 mg/L, <0.02-0.18 mg/L and 2.05-10.42 mg/L in group A, B and C, respectively (Table 2). The UCysC:C varied from 0·01 to 0·32 in group A, 0·1 to 0·11 in group B and 3·43 to 26·45 in group C (Table 2). Serum creatinine of dogs with renal disease ranged between 183 and 1379 μmol/L (reference interval: 36 to 120 μmol/L) and the range for urea was 13·3 to 100·9 mmol/L (reference interval: 3·3 to 8·0 mmol/L). The distribution of SCysC in dogs with renal disease was significantly higher than in healthy dogs and dogs with non-renal disease (Kruskal-Wallis test; P<0·001). The same result was obtained when the distribution of UCysC and UCysC:C were compared between the three cohorts (-Kruskal-Wallis test; P<0·001) (Figs 1 and 2). No significant difference in the distribution of SCysC and UCysC was found between groups A and B (Mann-Whitney U test; P=0·87 and P=0·20, respectively). A statistical difference between these two groups was found for the distribution of UCysC:C (-Mann-Whitney U test; P=0·004) (Fig 3).

Table 2. Urinary CysC and UCysC:C in groups A, B and C (A: healthy dogs, B: non-renal disease dogs and C: renal disease dogs)
 ABC
  1. CysC, urinary cystatin C; UCysC:C, urinary cystatin C to creatinine ratio; sd, standard deviation. ease: group C)

Urinary cystatin C
n141715
Mean (mg/L)0·050·065·28
Median (mg/L)0·020·054·43
sd (mg/L)0·060·042·75
Minimum (mg/L)0·020·022·05
Maximum (mg/L)0·260·1810·42
Urinary cystatin C to creatinine ratio
n141715
Mean (mg/L)0·040·0411·47
Median (mg/L)0·010·0410·87
sd (mg/L)0·080·037·31
Minimum (mg/L)0·010·013·43
Maximum (mg/L)0·320·1126·45
image

Figure 1. Urinary cystatin C concentrations in renal disease dogs, healthy dogs and dogs with non-renal disease. There is a statistical difference between the three cohorts (Kruskal-Wallis test; P<0·001). The top of the boxes represents the 75th percentile, the bottom of the boxes represents the 25th percentile. The lines in the middle represent the 50th percentile. The empty circle represents values that are between 1·5 and 3 times the interquartile range. The stars represent values exceeding more than thrice the interquartile range

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image

Figure 2. Urinary cystatin C to creatinine ratio in renal disease dogs, healthy dogs and dogs with non-renal disease. There is a statistical difference between the three cohorts (Kruskal-Wallis test; P<0·001). The empty circle represents values that are between 1·5 and 3 times the interquartile range. The stars represent values exceeding more than thrice the interquartile range

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image

Figure 3. Urinary cystatin C to creatinine ratio in healthy dogs and dogs with non-renal disease. There is a statistical difference between the two control groups (Mann-Whitney U test; P=0·004). The empty circle represents values that are between 1·5 and 3 times the interquartile range. The stars represent values exceeding more than thrice the interquartile range

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Three dogs in group A had values of UCysC exceeding more than thrice the interquartile range of distribution (extreme values). After normalisation of the cystatin C with the urinary creatinine, only two of these dogs still had an extreme value (Fig 3).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The primary aim of the study was to validate the PETIA for the measurement of UCysC in dogs. The assay is precise, linear and rapid. The recovery test showed that PETIA is accurate for measuring human cystatin C when diluted in canine urine. This only proves that there are no matrix effects on this molecule but does not confirm the accuracy of this method for evaluation of canine UCysC. This is a limitation of the study and was unavoidable because canine cystatin C is currently not commercially available. During the validation study, the LoB was evaluated but the limit of quantification was not verified because low values of cystatin C are not clinically significant.

Second, this preliminary study suggests that UCysC and UCysC:C are able to discriminate dogs with renal disease from dogs without renal disease. As cystatin C is reabsorbed and degraded by the proximal tubular cells, it is plausible to suggest that UCysC:C is a promising marker for evaluating renal tubular function in dogs. Urinary CysC:C would not therefore replace the measurement of serum creatinine or cystatin C, but would be considered a complementary test able to investigate tubular rather than glomerular function. This hypothesis is in agreement with the conclusion of Conti and others (2006), who showed that UCysC accurately detects tubular dysfunction among pure and mixed nephropathies in people. Although there are no studies in veterinary medicine that describe the kinetics of cystatin C in dogs, the findings of this report support a pathway similar to humans.

This study confirmed, as reported previously in human and veterinary medicine, that SCysC concentration is independent of gender but correlates with age in dogs without renal disease (Braun and others 2002). This correlation was also described in human where a significant elevation of SCysC was found in patients older than 60 years probably due to physiological ageing of renal function (Galteau and others 2001). Moreover, this study has shown that SCysC and UCysC do not differ significantly between healthy dogs and dogs affected by non-renal disease but UCysC:C was statistically different between these two groups. The reason for this finding was not investigated further because of limited clinical relevance but it is believed that the low numbers of animals included in each cohort or the different median age of the two groups may have contributed to the result.

Three clinically healthy dogs (group A) had values of UCysC exceeding thrice the interquartile range. After normalisation of the cystatin C with the urinary creatinine, only two of these dogs still had an extreme value. These cases were an eight-year-old male dog and a one-year-old male dog admitted for castration and there were no signs of renal disease, but follow-up information is lacking. The low number of dogs included in group A or a skewed distribution of this analyte within the population may have contributed. Alternatively, these dogs could reflect true outliers or might have been misclassified as healthy. However, their UCysC:C was still significantly lower than the range of UCysC:C seen in renal patients.

This study has several limitations, the main one being the lack of follow-up information for the majority of the patients, precluding the assessment of the potential of UCysC:C as a prognostic marker. Moreover, because only dogs with overt signs of renal disease were recruited into group C, this overlooked the possible ability of this marker to identify early kidney disease. Identification of renal disease is important in dogs, but early diagnosis remains difficult. Further studies should be directed towards investigating this new marker to detect early renal disease because currently there are no sensitive indirect markers of early subclinical renal disease.

A few papers in human medicine have shown that there is a correlation between the UCysC concentration and the degree of proteinuria. It has been postulated that albumin and cystatin C compete for the same receptors on the luminal face of the tubular cells; therefore a competitive inhibition of the UCysC reabsorption may occur, especially if the degree of albuminuria is severe (Thielemans and others 1994, Tkaczyk and others 2004). In this article, the influence that proteinuria might have on the cystatin C was not investigated.

Finally, the number of dogs evaluated was low, limiting the potential significance of the findings. A larger number of dogs should be recruited to increase the power of the study.

These preliminary results indicate that this marker is able to discriminate dogs with or without renal disease, justifying the continuation of the study. Further investigations would be needed to verify if the degree of UCysC:C is proportional to the severity of the tubular damage and its prognostic value. Moreover, comparison of UCysC with other markers of tubulopathy should also be investigated (e.g. retinol binding protein).

In conclusion, despite the limitations of this study, these preliminary results show that measurement of UCysC appears to be a novel marker for renal disease and likely specific for tubular function. The good performance of the PETIA allows a rapid turnaround and provides an easy and non-invasive way to assess renal function in dogs. All the characteristics, including the non-invasiveness of sample collection, the rapid turnaround of the results, the organ specificity and the cost effectiveness of the test, if performed on a large scale, would fulfil most of the criteria for being defined as a reasonable renal biomarker.

Acknowledgements

This work was funded by the RCVS Trust, Petsavers and Cambridge Infectious Diseases Consortium (CIDC). The authors would like to acknowledge Stacey Davey and Miranda Garfoot for helping in the validation of the PETIA; Michela Corte and Andrea Galvagni for helping recruiting the cases and Animal Health Trust, PTDS and CTDS Laboratories for submitting the samples, and Beckman Coulter for providing the assay kit at a discounted price.

Conflict of interest

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

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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