Liesbeth Ghys

Routine Health Screening: Findings in apparently healthy middle-aged and old cats

Study rationale: Veterinary practitioners often perform geriatric health screening in cats. Unfortunately, scientific information regarding clinical and laboratory abnormalities and normal blood pressure values in elderly cats is scarce. This prospective study evaluated routine health screening tests in apparently healthy middle-aged and old cats. Protocol: One hundred cats of 6 years and older underwent blood pressure measurement, physical examination, blood and urine analysis, indirect fundoscopy and bilateral Schirmer tear tests. Findings: Mean systolic blood pressure (SBP) was 133.6 ± 21.5 mmHg. Increased SBP (>160 mmHg) was observed in eight cats, submandibular lymphadenopathy in 32, gingivitis in 72, heart murmur in 11, thyroid goitre in 20, increased creatinine in 29, hyperglycaemia in 25, increased total thyroxine in three, feline immunodeficiency virus positivity in 14, crystalluria in 41, borderline proteinuria in 25 and overt proteinuria in two. Mean tear production was very similar for both eyes and none of the cats had ocular lesions secondary to hypertension. Clinical significance: Old cats (>10 years) had significantly higher SBP, heart rate, murmur frequency, thrombocyte count, urine protein:creatinine ratio and serum urea and bilirubin concentrations, and significantly lower body condition score, haematocrit, albumin and total calcium concentrations than middle-aged cats (6–10 years). The common occurrence of physical examination and laboratory abnormalities in apparently healthy old cats underlines the need for regular health checks and the development of age-dependent laboratory reference intervals.

Cystatin C: A New Renal Marker and Its Potential Use in Small Animal Medicine

The occurrence of chronic kidney disease is underestimated in both human and veterinary medicine. Glomerular filtration rate (GFR) is considered the gold standard for evaluating kidney function. However, GFR assessment is time‐consuming and labor‐intensive and therefore not routinely used in practice. The commonly used indirect GFR markers, serum creatinine (sCr) and urea, are not sufficiently sensitive or specific to detect early renal dysfunction. Serum cystatin C (sCysC), a proteinase inhibitor, has most of the properties required for an endogenous GFR marker. In human medicine, numerous studies have evaluated its potential use as a GFR marker in several populations. In veterinary medicine, this marker is gaining interest. The measurement is easy, which makes it an interesting parameter for clinical use. This review summarizes current knowledge about cystatin C (CysC) in humans, dogs, and cats, including its history, assays, relationship with GFR, and biological and clinical variations in both human and veterinary medicine.

Biological validation of feline serum cystatin C: The effect of breed, age and sex and establishment of a reference interval

Chronic kidney disease (CKD) is common in cats, but the routine renal markers, serum creatinine (sCr) and urea, are not sensitive or specific enough to detect early CKD. Serum cystatin C (sCysC) has advantages over sCr, both in humans and dogs, and sCysC concentration is significantly higher in cats with CKD than in healthy cats. The objective of this study was to determine the effect of age, sex and breed on feline sCysC and to establish a reference interval for feline sCysC. In total, 130 healthy cats aged 1-16 years were included. sCysC was determined using a validated particle-enhanced nephelometric immunoassay. sCr, urea, urine specific gravity, urinary protein:creatinine ratio (UPC) and systolic blood pressure (SBP) were also measured. No significant differences in sCysC concentration were observed among young, middle-aged and geriatric cats, female intact, female neutered cats, male intact and male neutered cats, or among purebred and domestic short-or longhaired cats. The 95% reference interval for feline sCysC was determined to be 0.58-1.95 mg/L. sCr was significantly higher in geriatric cats than young cats. Serum urea in geriatric cats was significantly higher than in middle-aged and young cats (P = 0.004 and P <0.001, respectively). SBP in geriatric cats was significantly higher than in both middle-aged and young cats (P = 0.004 and P = 0.040, respectively). Male neutered and female neutered cats had significantly higher serum urea concentrations than female intact cats (P = 0.003 and P = 0.006, respectively). Male intact cats had a significantly higher UPC than female intact and female neutered cats (P = 0.02 for each comparison). There were no significant differences among sex groups for USG. It is of concern that sCysC in the majority of cats with CKD in previous studies falls within the reference interval calculated in this study. Further studies are warranted to evaluate the diagnostic value of sCysC as a renal marker in cats.

Analytical validation of a human particle-enhanced nephelometric assay for cystatin C measurement in feline serum and urine

BACKGROUND In people and dogs, Cystatin C (CysC), a renal glomerular and tubular marker, seems superior to serum creatinine to estimate the glomerular filtration rate (GFR). A particle-enhanced nephelometric immunoassay is available to measure human CysC, but there are no reports in cats. OBJECTIVE The goal of this study was the validation of the human CysC nephelometric assay with feline serum and urine, and to perform a pilot study comparing serum and urine CysC between healthy cats and cats with chronic kidney disease (CKD). METHODS Western blot analysis was used to assess cross-reactivity between the polyclonal rabbit anti-human CysC antibody and feline CysC. Imprecision and linearity were determined for feline serum and urine CysC. Serum and urine CysC were measured in 10 healthy and 10 CKD cats. RESULTS Cross-reactivity between the polyclonal rabbit anti-human CysC antibody and feline CysC was demonstrated. Intra- and inter-assay coefficients of variation in feline serum and urine were 1.3% and 0.4%, and 12.5%, and 4.1%, respectively. Cats with CKD had a significantly higher serum CysC concentration (1.24 [0.63-2.99] vs 0.79 [0.43-1.05] mg/L; P = .02) and urine CysC/urinary Creatinine (uCr) ratio (565.6 [0-1311] vs < 0.049/uCr mg/mol; P = .005) compared with healthy cats. CONCLUSIONS The human nephelometric assay showed satisfactory validation results for feline CysC. Cats with CKD had a significantly higher sCysC concentration and uCysC/uCr ratio compared with healthy cats. Additional studies are necessary to evaluate CysC as an early marker of renal damage in cats.

Long-term follow-up of renal function assessing serum cystatin C in dogs with diabetes mellitus or hyperadrenocorticism.

BACKGROUND Serum cystatin C (sCysC) is used as biomarker for glomerular filtration rate (GFR). The effects of diabetes mellitus (DM) on renal function in dogs are unclear. Some renal variables have been evaluated in dogs with hyperadrenocorticism (HAC), but not sCysC. OBJECTIVES The purpose of this study was the validation of a particle-enhanced nephelometric immunoassay (PENIA) for measuring canine sCysC, and to assess renal function in dogs with DM or HAC. METHODS A PENIA was analytically validated for canine sCysC by determining imprecision and linearity. In a longitudinal 6-month study, renal function of 14 DM dogs was assessed, using serum creatinine, GFR, urinary protein-to-creatinine (UPC) ratio, urinary markers, systolic blood pressure (SBP), and sCysC, and compared to 17 healthy dogs at baseline. Furthermore, sCysC was measured at initial presentation and during a 12-month follow-up in 22 HAC dogs. RESULTS The sCysC intra- and inter-assay variation coefficients were < 8% and highly linear (r = .997). About 33% and 67% of DM dogs had persistent proteinuria and systemic hypertension, respectively, but there were no significant differences in GFR, UPC, and urinary markers over time, and compared with healthy dogs at initial presentation. Serum CysC decreased significantly (P < .05) over time within the DM group. It did not change significantly over time within the HAC group. CONCLUSIONS A PENIA measured sCysC linearly and precisely. There were no clinically relevant renal alterations over time in dogs with DM, although persistent proteinuria was observed. In dogs with HAC, sCysC measurement was not useful, although significant GFR changes occurred over time.

Evaluation of Cystatin C for the Detection of Chronic Kidney Disease in Cats

Background Serum cystatin C (sCysC) and urinary cystatin C (uCysC) are potential biomarkers for early detection of chronic kidney disease (CKD) in cats. An in‐depth clinical validation is required. Objectives To evaluate CysC as a marker for CKD in cats and to compare assay performance of the turbidimetric assay (PETIA) with the previously validated nephelometric assay (PENIA). Animals Ninety cats were included: 49 CKD and 41 healthy cats. Methods Serum CysC and uCysC concentrations were prospectively evaluated in cats with CKD and healthy cats. Based on plasma exo‐iohexol clearance test (PexICT), sCysC was evaluated to distinguish normal, borderline, and low GFR. Sensitivity and specificity to detect PexICT < 1.7 mL/min/kg were calculated. Serum CysC results of PENIA and PETIA were correlated with GFR. Statistical analysis was performed using general linear modeling. Results Cats with CKD had significantly higher mean ± SD sCysC (1.4 ± 0.5 mg/L) (P < .001) and uCysC/urinary creatinine (uCr) (291 ± 411 mg/mol) (P < .001) compared to healthy cats (sCysC 1.0 ± 0.3 and uCysC/uCr 0.32 ± 0.97). UCysC was detected in 35/49 CKD cats. R 2 values between GFR and sCysC or sCr were 0.39 and 0.71, respectively (sCysC or sCr = μ + GFR + ε). Sensitivity and specificity were 22 and 100% for sCysC and 83 and 93% for sCr. Serum CysC could not distinguish healthy from CKD cats, nor normal from borderline or low GFR, in contrast with sCr. Conclusion Serum CysC is not a reliable marker of reduced GFR in cats and uCysC could not be detected in all CKD cats.

Serum and urinary cystatin C in cats with feline immunodeficiency virus infection and cats with hyperthyroidism.

Objectives The objective of this study was to investigate serum cystatin C (sCysC) and urinary cystatin C (uCysC) in cats with hyperthyroidism and cats with feline immunodeficiency virus (FIV). Methods Thirty cats with FIV, 26 hyperthyroid cats and 28 healthy cats were included. sCysC and uCysC:creatinine (uCysC/uCr) ratio were measured with a human particle-enhanced nephelometric immunoassay, previously validated for feline CysC measurement. Routine renal variables (serum creatinine [sCr], urine specific gravity, urinary protein:creatinine ratio [UPC]) were also measured in the three groups. Results Cats with hyperthyroidism had significantly higher sCysC and higher uCysC/uCr ratio, lower sCr and a higher UPC than healthy cats. Cats with FIV infection did not show a significantly higher sCysC concentration but had a significantly higher sCr and UPC than healthy cats. uCysC could be detected in only four of them. Conclusions and relevance This study demonstrated that sCysC is increased in cats with hyperthyroidism, in contrast with sCr, but not in cats with FIV. Many hyperthyroid cats, but only four cats with FIV, had an elevated uCysC/uCr ratio. Further studies may reveal if uCysC might be a valuable marker for tubular dysfunction in cats.

Routine kidney variables, glomerular filtration rate and urinary cystatin C in cats with diabetes mellitus, cats with chronic kidney disease and healthy cats

Objectives Diabetic kidney disease (DKD) is a frequent and serious complication in human diabetic patients, but data are limited in cats. This study was undertaken to assess whether diabetic cats are susceptible to DKD. Methods Kidney function was compared between 36 cats with diabetes mellitus (DM), 10 cats with chronic kidney disease (CKD) and 10 age-matched healthy cats by measuring routine kidney variables (serum creatinine [sCreat], serum urea [sUrea], urine specific gravity [USG], urinary protein:creatinine ratio [UPC]), urinary cystatin C:creatinine ratio and glomerular filtration rate (GFR). Urinary cystatin C (uCysC) was measured with a human particle-enhanced nephelometric immunoassay, validated to measure feline cystatin C, in all but two diabetic cats. GFR was evaluated by exo-iohexol clearance in 17 diabetic cats, all cats with CKD and all healthy cats. Results Diabetic cats had significantly (mean ± SD) lower sCreat (123 ± 38 vs 243 ± 80 µmol/l), sUrea (11 ± 3 vs 18 ± 7 mmol/l) and urinary cystatin C:creatinine ratio (6 ± 31 vs 173 ± 242 mg/mol), and a significantly higher USG (1.033 ± 0.012 vs 1.018 ± 0.006) and GFR (2.0 ± 0.7 vs 0.8 ± 0.3 ml/min/kg) compared with cats with CKD. Compared with healthy cats, diabetic cats only had significantly lower USG (1.033 ± 0.012 vs 1.046 ± 0.008). Proteinuria (UPC >0.4) was present in 39% of diabetic cats, in 30% of cats with CKD and in none of the healthy cats. However, the UPC did not differ statistically between the three groups. Conclusions and relevance Based on evaluation of routine kidney variables, GFR and uCysC as a tubular marker at a single time point, a major impact of feline DM on kidney function could not be demonstrated.

The effect of feeding, storage and anticoagulant on feline serum cystatin C

Serum cystatin C (sCysC) is a possible marker for early detection of chronic kidney disease (CKD) in cats. In contrast with serum creatinine (sCr), feline sCysC is not affected by age, breed or sex. However, further biological and clinical validation is required. The objectives of this study were: (1) to investigate if food intake and circadian rhythm affect feline sCysC; (2) to determine the stability of sCysC under different storage conditions, and (3) to investigate if plasma concentrations of CysC (pCysC) differed from sCysC. A crossover study with 10 healthy laboratory cats fed the same commercial dry food was performed to study the influence of feeding and diurnal variation. Storage effects and comparison of pCysC with sCysC were determined using healthy cats (n = 3 and n = 10, respectively) and cats with CKD (n= 4 and n = 17, respectively). A significant daily sCysC variation was seen. Pre- and postprandial sCysC and sCr concentrations did not change significantly. Serum CysC significantly increased during storage at room temperature. After freezing, sCysC significantly decreased after 5 and 12 months at both -20 °C and -72 °C. Plasma CysC was significantly lower than sCysC. These findings suggest that it is not mandatory to fast cats before evaluation of sCysC and sCr. Samples were stable during routinely used storage conditions. Based on these findings, freezing for more than 5 months is not recommended, although additional studies are required to evaluate the clinical relevance of decreased sCysC after prolonged storage. Plasma and serum CysC cannot be compared directly.

Systolic blood pressure, routine kidney variables and renal ultrasonographic findings in cats naturally infected with feline immunodeficiency virus

Objectives Hypertension is a common cause of proteinuria in HIV-infected people. In cats, feline immunodeficiency virus (FIV) infection appears to be associated with proteinuria. Therefore, the results from systolic blood pressure (SBP) measurements in naturally infected FIV-positive cats were reviewed to assess whether hypertension contributes to the observed proteinuria in these cats. Ultrasonographic findings in FIV-positive cats were reviewed to complete renal assessment and to extend the scant knowledge on renal ultrasonography in cats. Methods Data from client-owned, naturally infected FIV-positive cats were retrospectively reviewed. To obtain a control group, records were reviewed from age-matched, privately owned, FIV-negative cats. Results Data from 91 FIV-infected and 113 control cats were compared. FIV-infected cats showed a significantly lower SBP (P <0.0001) and significantly fewer FIV-infected cats were hypertensive (⩾160 mmHg) compared with control cats (P = 0.025). The prevalence of renal azotaemia did not significantly differ between groups, although FIV-infected cats had significantly lower urine specific gravity (USG) (P = 0.0273) and a higher incidence of USG below 1.035 (P = 0.043). Urinary protein:creatinine ratio (UPC) was significantly higher in FIV-infected cats (P = 0.0005) and proteinuria (UPC >0.4) occurred more frequently in FIV-infected cats (P <0.001). Renal ultrasonography showed abnormalities in 60/91 FIV-infected cats, with hyperechogenic cortices in 39/91 and enlarged kidneys in 31/91. Conclusions and relevance Hypertension can be excluded as a common cause of renal damage leading to proteinuria in FIV-infected cats. Proteinuria and poorly concentrated urine are common in naturally infected FIV-positive cats, in contrast to azotaemia. Clinicians should cautiously interpret ultrasonographic abnormalities as these occur in over half of FIV-infected cats.