• View in gallery
    Figure 1—

    Scatterplots of UC/C data for client-owned geriatric (≥ 9 years of age) cats classified according to their magnitude of proteinuria (nonproteinuric, UP/C < 0.2 [n = 114]; borderline proteinuric, UP/C = 0.2 to 0.4 [40]; proteinuric, UP/C > 0.4 [34]). Horizontal lines indicate median values. *Values differ significantly (P < 0.05) between indicated groups on ends of brackets.

  • View in gallery
    Figure 2—

    Scatterplots of UC/C data obtained at enrollment of client-owned geriatric (≥ 9 years of age) cats without (n = 19) and with (61) azotemia at their 12-month reexamination. See Figure 1 for key.

  • 1.

    Miyazaki MKamiie KSoeta S, et al.Molecular cloning and characterization of a novel carboxylesterase-like protein that is physiologically present at high concentrations in the urine of domestic cats (Felis catus). Biochem J 2003;370:101110.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Holmes RSCox LAVandeberg JL. Mammalian carboxylesterase 5: comparative biochemistry and genomics. Comp Biochem Physiol Part D Genomics Proteomics 2008;3:195204.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Miyazaki MYamashita THosokawa M, et al.Species-, sex-, and age-dependent urinary excretion of cauxin, a mammalian carboxylesterase. Comp Biochem Physiol B Biochem Mol Biol 2006;145:270277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Miyazaki MYamashita TSuzuki Y, et al.A major urinary protein of the domestic cat regulates the production of felinine, a putative pheromone precursor. Chem Biol 2006;13:10711079.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Miyazaki MYamashita TTaira H, et al.The biological function of cauxin, a major urinary protein of the domestic cat (Felis catus). In: Hurst JLBeynon RJRoberts SC, et al, eds. Chemical signals in vertebrates 11. New York: Springer, 2008;5160.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lulich JPOsborne CAO'Brien TD, et al.Feline renal failure: questions, answers, questions. Compend Contin Educ Pract Vet 1992;14:127152.

    • Search Google Scholar
    • Export Citation
  • 7.

    Ross SJOsborne CAKirk CA, et al.Clinical evaluation of dietary modification for treatment of spontaneous chronic kidney disease in cats. J Am Vet Med Assoc 2006;229:949957.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Miyazaki MSoeta SYamagishi N, et al.Tubulointerstitial nephritis causes decreased renal expression and urinary excretion of cauxin, a major urinary protein of the domestic cat. Res Vet Sci 2007;82:7679.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Elliott J. Staging chronic kidney disease. In: Elliott JGrauer GF, eds. BSAVA manual of canine and feline nephrology and urology. 2nd ed. Kundli Haryana, India: Replika Press Pvt Ltd, 2007;163.

    • Search Google Scholar
    • Export Citation
  • 10.

    Jepson REBrodbelt DVallance C, et al.Evaluation of predictors of the development of azotemia in cats. J Vet Intern Med 2009;23:806813.

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    • Search Google Scholar
    • Export Citation

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Measurement of urinary cauxin in geriatric cats with variable plasma creatinine concentrations and proteinuria and evaluation of urine cauxin-to-creatinine concentration ratio as a predictor of developing azotemia

Rosanne E. JepsonDepartment of Veterinary Clinical Science, Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, England.

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Harriet M. SymeDepartment of Veterinary Clinical Science, Royal Veterinary College, North Mymms, Hatfield, Hertfordshire AL9 7TA, England.

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Peter MarkwellWALTHAM, a Division of Mars Incorporated, Freeby Ln, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire LE14 4RS, England.

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Masao MiyazakiInstitute of Glycoscience, Tokai University, 1117 Kitakaname, Hiratsuka-shi Kanagawa, 259-1292, Japan.

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Tetsuro YamashitaDepartment of Agro-Bioscience, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, 020-8550, Japan.

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Jonathan ElliottVeterinary Basic Science, Royal Veterinary College, Camden, London NW1 0TU, England.

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Abstract

Objective—To evaluate urine cauxin immunoreactivity in geriatric cats with variable plasma creatinine concentrations and proteinuria and to assess urinary cauxin-to-creatinine concentration ratio (UC/C) as a predictor of developing azotemia.

Animals—188 client-owned geriatric (≥ 9 years of age) cats.

Procedures—A direct immunoassay was developed and validated for the quantification of urinary cauxin relative to a standard curve generated from a urine sample with high cauxin immunoreactivity. Relationships among UC/C, plasma creatinine concentration, and proteinuria were assessed. Nonazotemic cats were recruited and followed for 12 months. Urinary cauxin-to-creatinine concentration ratio was evaluated as a predictor of development of azotemia in these cats.

Results—No relationship was evident between UC/C and plasma creatinine concentration. A weak positive correlation was identified between UC/C and urine protein-to-creatinine concentration ratio (r = 0.212). At entry to the longitudinal study, those cats that later developed azotemia had a UC/C that was significantly higher than in those remaining nonazotemic after 12 months.

Conclusions and Clinical Relevance—The UC/C did not vary with severity of azotemia but appeared contributory to the feline urinary proteome. High UC/C values were predictive of the geriatric cats in our study developing azotemia. However, it seems unlikely that UC/C will provide additional information about the measurement of urine protein-to-creatinine concentration ratio as a biomarker for the development of azotemia in cats.

Abstract

Objective—To evaluate urine cauxin immunoreactivity in geriatric cats with variable plasma creatinine concentrations and proteinuria and to assess urinary cauxin-to-creatinine concentration ratio (UC/C) as a predictor of developing azotemia.

Animals—188 client-owned geriatric (≥ 9 years of age) cats.

Procedures—A direct immunoassay was developed and validated for the quantification of urinary cauxin relative to a standard curve generated from a urine sample with high cauxin immunoreactivity. Relationships among UC/C, plasma creatinine concentration, and proteinuria were assessed. Nonazotemic cats were recruited and followed for 12 months. Urinary cauxin-to-creatinine concentration ratio was evaluated as a predictor of development of azotemia in these cats.

Results—No relationship was evident between UC/C and plasma creatinine concentration. A weak positive correlation was identified between UC/C and urine protein-to-creatinine concentration ratio (r = 0.212). At entry to the longitudinal study, those cats that later developed azotemia had a UC/C that was significantly higher than in those remaining nonazotemic after 12 months.

Conclusions and Clinical Relevance—The UC/C did not vary with severity of azotemia but appeared contributory to the feline urinary proteome. High UC/C values were predictive of the geriatric cats in our study developing azotemia. However, it seems unlikely that UC/C will provide additional information about the measurement of urine protein-to-creatinine concentration ratio as a biomarker for the development of azotemia in cats.

Cauxin is a 70-kDa carboxylesterase capable of hydrolyzing aromatic and aliphatic substrates containing ester, thioester, and amide bonds and is a major component of the feline urinary proteome.1,2 Use of northern blotting techniques has revealed cauxin mRNA within feline urine and kidney tissue but not in serum or other tissues.1 Immunohistochemical assays have identified colocalization of cauxin with megalin in the proximal straight tubules from where it is secreted into the urine.3 Excretion of cauxin appears to be age dependent, with no cauxin evident in the urine of cats < 3 months of age when immunoblotting techniques are used.3,4 Urine and kidney tissue from sexually intact male cats have higher cauxin concentrations than similar tissue from neutered male and sexually intact or spayed female cats.3 Although the biological importance of cauxin has not been fully elucidated, cauxin hydrolyses 3-methylbutanol-cysteinylglycine to the feline pheromone felinine and to glycine. It is therefore suggested that cauxin may play an important role in behavioral territorial marking in cats.4,5

Chronic kidney disease is commonly recognized in geriatric cats.6,7 In 1 study,8 immunohistochemical techniques revealed fewer proximal straight tubules with evidence of cauxin staining in kidneys of cats with a range of mild to severe azotemia and varying degrees of tubulointerstitial nephritis (n = 15) than in young healthy control cats (5). Cauxin was also identified in the urine of the healthy cats and in 2 cats with mild tubulointerstitial nephritis. However, urinary cauxin was not detected in 4 cats with severe tubulointerstitial nephritis and azotemia. It was therefore hypothesized that a reduction in cauxin activity or concentration within feline urine may be a potential biomarker of early cell damage in renal proximal tubules. Such a marker would have considerable clinical impact on the early diagnosis of CKD in cats.

The purpose of the study reported here was to develop and validate a direct immunoassay for the quantification of urinary cauxin; to evaluate the relationships among UC/C, plasma creatinine concentration, and proteinuria; and to examine the use of UC/C as a predictor of the development of azotemia.

Materials and Methods

Cats—Cats (≥ 9 years of age) were recruited from 2 geriatric feline clinics held at general practices in central London (Beaumont Animals' Hospital, Royal Veterinary College, Camden, London; and Peoples Dispensary for Sick Animals, Bow, London). Cats with previously diagnosed metabolic or clinically important cardiovascular disease or those receiving any long-term medications other than parasiticides were excluded from the study. At entry to the study, a full history was obtained from owners, and all cats received a physical examination and measurement of SAP by use of the Doppler technique.a The study protocol was approved by the Ethics and Welfare Committee of the Royal Veterinary College, and consent was obtained from all cat owners.

Sample collection—Owners were routinely asked to withhold food from cats for 8 hours prior to visiting the practice. Blood samples were collected via jugular venipuncture into tubes containing lithium heparin. Tubes were subsequently centrifugedb at 1,000 × g at 4°C for 10 minutes to yield heparinized plasma, which was used to perform a full biochemical analysis and assessment of total thyroxine concentration at an external commercial laboratory.c

Urine samples were collected from all cats via cystocentesis and were chilled on ice between collection and analysis. Urinalysis was performed within 6 hours after sample collection and included measurement of specific gravity by refractometry, measurement of pH,d semiquantitative biochemical analysis with chemical reagent strips,e and examination of urine sediment. After urinalysis, urine samples were centrifuged at 1,000 × g at 4°C for 10 minutes; urine supernatant was then separated and stored at −80°C. The UP/C in stored samples was assessed at an external laboratory at which urine protein concentration was measured by use of a colorimetric pyrogallol red method and urine creatinine concentration was measured by use of a colorimetric picric acid method. Urine samples were excluded from analysis when there was evidence of a urinary tract infection, bactiuria, pyuria, or gross hematuria.

Direct immunoassay for urinary cauxin—A direct immunoassay was developed to enable relative quantification of feline urinary cauxin. Because no purified or synthetic cauxin was commercially available, a relative standard was generated by use of urine pooled from 5 sexually intact male cats. Sexually intact male cats reportedly have substantially higher urinary concentrations of cauxin than their neutered counterparts or female cats.3 An immunoblot study was performed, as described by Miyazaki et al,8 and revealed a strong electrophoretic band of approximately 70 kDa, consistent with the presence of cauxin. Serial dilutions (1:100 to 1:3,200) of the pooled sample were used to generate a standard curve from which the relative concentration of cauxin within the test samples could be interpolated. The pooled urine from the sexually intact male urine was aliquoted and stored at −20°C such that it was not subjected to multiple freeze-thaw cycles.

Flat-bottomed polystyrene platesf were coated overnight with standard or sample (100 ML/well). All standard and sample dilutions were prepared with TBSST and 0.5% bovine serum albumin.g Typically, urine samples from geriatric cats were diluted between 5 and 200-fold so absorbance values would fall on the linear region of the standard curve when plotted. All incubations were carried out at room temperature (approx 21°C) within a humidified environment. The wells were washed 4 times with 400 ML of wash buffer (TBSST)/well, blocked for 1 hour with TBS and 0.5% bovine serum albumin (100 ML/well), and washed again as previously described.

A rabbit polyclonal antibody that was raised against a 20–amino acid peptide sequence at the C-terminus of the cauxin protein was diluted with sample diluent (1:2,000), and 100 ML/well was loaded.8 Following 1 hour of incubation, an additional wash step was performed, and horseradish peroxidase–conjugated secondary antibodyh diluted with sample diluent (1:2,000) was loaded (100 ML/well). A final wash step was performed to enable removal of any unbound conjugated antibody prior to application of 3,3a,5,5a-tetramethylbenzidinei (100 ML/well). Color was allowed to develop for 5 minutes, and the reaction was stopped by application of 1M H2SO4 (50 ML/well).g The absorbance was measured at 450 nm with a spectrophotometer.j All samples and standards were run in duplicate.

With no known standard available, the amount of cauxin in urine samples was expressed as relative immunoreactivity. This relative cauxin immunoreactivity was then standardized to urinary creatinine concentration as UC/C to accommodate for variation in urine volume.

Assay development and validation—The optimum dilution factors for the primary and secondary antibody were evaluated by repeating assessment of the relative standard curve with dilutions from 1:1,000 to 1:10,000. The lowest antibody concentration yielding maximal absorption was chosen for use within the immunoassay. Assay validation was performed with urine samples that had been stored at −80°C. Interassay and intra-assay variability were evaluated as indicators of assay performance. The analytic sensitivity of the assay was calculated from the mean interpolated relative cauxin immunoreactivity corresponding to the mean ± SD of the zero standard. The lower limit of detection for the assay was taken as the lowest sample dilution that provided an intra-assay CV of < 20%. Dilutional parallelism was achieved for a single urine sample with high cauxin immunoreactivity. The effect of storage of urine samples on urinary cauxin immunoreactivity was investigated by assessing urine cauxin immunoreactivity in fresh urine samples that had been stored on ice for 4 to 6 hours and after 1 week of storage at 4°C and 1, 2, 6, and 12 weeks of storage at −20°C.

Evaluation of cauxin in cats—Stored urine samples from nonazotemic cats with no evidence of CKD and from cats in which CKD was previously diagnosed were selected. Cats were grouped according to their plasma creatinine concentration (I, < 1.6 mg/dL; IIa, 1.6 to 2.0 mg/dL; IIb, 2.0 to 2.8 mg/dL; III, 2.8 to 5.0 mg/dL; IV, > 5.0 mg/dL) and also according to magnitude of proteinuria (nonproteinuric, < 0.2; borderline proteinuric, 0.2 to 0.4; proteinuric, > 0.4). These categories mirrored those of the IRIS9; however, it should be appreciated that the study groups contained cats in which CKD had not previously been diagnosed. Relative urine cauxin immunoreactivity was measured with the direct immunoassay and expressed as UC/C.

Evaluation of cauxin as a predictor of azotemia— For entry to the prospective cohort study, cats were required to be nonazotemic with a plasma creatinine concentration < 2.0 mg/dL. Cats with plasma total thyroxine concentrations > 55 nmol/L were considered hyperthyroid and excluded from the study. Stored urine samples were used to assess urine cauxin immunoreactivity at entry to the study by use of the direct immunoassay as previously described, and results were expressed as UC/C.

Normotensive nonazotemic cats were invited for examinations after 6 and 12 months of enrollment. At each revisit, cats received a full physical examination, measurement of SAP, and full plasma biochemical analysis. Total thyroxine concentration was evaluated annually in all nonazotemic cats or in cats in which the history (eg, polyphagia or weight loss), clinical examination findings (eg, palpable goiter, tachycardia, or low body condition score), and biochemical analysis (eg, high alanine aminotransferase or alkaline phosphatase activity) were consistent with a potential diagnosis of hyperthyroidism. When a bladder was palpable, urine samples were obtained at each subsequent visit via cystocentesis and analyzed as previously described. Nonazotemic cats with systemic hypertension were treated with amlodipine besylatek (0.625 to 1.25 mg/cat/d), and after stabilization of arterial blood pressure, owners were offered the opportunity to have their cats reexamined every 8 weeks. In these cats, plasma biochemical analysis and, when possible, urinalyses were performed on alternate visits. The renal status of cats was assessed at the 12-month point, and cats were classified as remaining nonazotemic (plasma creatinine concentration < 2.0 mg/dL) or having developed azotemia (plasma creatinine concentration ≥ 2.0 mg/dL). Cats that were not returned for reexamination, despite multiple invitations, were considered lost to follow-up.

Statistical analysis—Statistical analysis was performed by use of computer software.l,m Unless otherwise stated, data are presented as median (25th, 75th percentile), and a value of P < 0.05 was considered significant. The effect of sample handling on cauxin immunoreactivity was evaluated with a nonparametric Friedman test and a Dunn multiple comparisons post hoc analysis. Data for urine cauxin immunoreactivity, UC/C, UC/UP, and UP/C were assessed for normal distributions via the Kolmogarov-Smirnov test and were logarithmically transformed for parametric statistical analysis.

The independent Student t test was used to compare logarithmically transformed UC/C data between neutered male and neutered female cats. A 1-way ANOVA was used to compare clinical data and logarithmically transformed UC/UP and logUC/C values between cats grouped by plasma creatinine concentration or proteinuria. When necessary, a Bonferroni post hoc adjustment was applied. Pearson correlation coefficients were calculated to evaluate the relationship between logarithmically transformed UP/C and logUC/C values. The independent Student t test was used to compare logUC/C values and clinical data at entry to the cohort study between those cats that remained nonazotemic and those that had developed azotemia by 12 months after enrollment.

Results

Cats—Ten cats (5 neutered males and 5 neutered females) were included in the diagnostic test evaluation study. The mean ± SD age was 12.9 ± 5.3 years, and the mean body weight was 3.9 ± 0.68 kg (n = 9).

For the prospective cohort study, 101 cats were enrolled, 10 of which were treated as idiopathic hypertensive. Eleven cats died within the 12-month follow-up, and a further 6 cats were lost to follow-up. Sixty-one cats remained nonazotemic, and 19 cats had developed azotemia by the 12-month point. Median (25th, 75th percentile) age of the cats that remained in the study at 12 months was 12 (11.0, 14.3) years for the nonazotemic cats and 13 (12.0, 15.0) years for the azotemic cats.

Validation of the direct immunoassay—Intraassay CVs (n = 6) for a urine sample with a low (mean, 433 arbitrary units) and high (mean, 2,283 arbitrary units) cauxin immunoreactivity were 5.6% and 6.8%; interassay CVs were 10.1% and 19.3%, respectively. Dilutional parallelism was evident. The calculated analytic sensitivity of the cauxin ELISA was 0.78 arbitrary U/mL. All urine samples examined had cauxin immunoreactivity much greater than the analytical sensitivity of the ELISA, requiring dilution at a ratio of at least 1:5 so that associated results would appear on the linear region of the standard curve when plotted.

In fresh urine samples (n = 5 cats), the median (25th, 75th percentile) cauxin immunoreactivity was quite low (19.8 [5.6, 22.6] arbitrary units); in 1 cat, it was not detectable. Urine cauxin immunoreactivity in these samples did not differ significantly after 7 days storage at 4°C (24.9 [11.4, 43.7]). Storage for 7 days at −20°C significantly increased the median urine cauxin immunoreactivity value from 19.8 (5.6, 22.6) to 169.0 (102.0, 401.0). There was no additional change in cauxin immunoreactivity in samples stored for 1 week or in those stored for 2, 6, and 12 weeks at −20°C.

Cauxin immunoreactivity and UC/C—Urine cauxin immunoreactivity and UC/C were evaluated in 188 cats with variable plasma creatinine concentration. No significant (P = 0.180) difference in logUC/C was identified between male (n = 95) and female (93) cats; therefore, data were combined for further statistical analysis. Clinical and urinary cauxin data from these cats, categorized according to plasma creatinine staging, were summarized (Table 1). Results of 1-way ANOVA indicated there was no significant (P = 0.164) difference in logUC/C values stratified by plasma creatinine concentration, although a significant difference in relative UC/UP was identified (P < 0.01). A significant (P < 0.001) difference in logUC/C was detected between groups with differing severity of proteinuria (Figure 1). A positive correlation was found between logUC/C and logarithmically transformed UP/C (r = 0.212; P < 0.001).

Table 1—

Median (25th, 75th percentile) values for clinical variables and urinary cauxin in 188 client-owned geriatric (≥ 9 years of age) cats with various ranges of plasma creatinine concentrations.*

VariablesPlasma creatinine concentration  
< 1.6 mg/dL (n = 51)1.6 to 2.0 mg/dL (n = 59)2.0 to 2.8 mg/dL (n = 38)
Creatinine (mg/dL)1.331.782.44
(1.24, 1.50)(1.66, 1.87)(2.19, 2.66)
Cauxin832.9a773.1a393.1b
(arbitrary units)(414.6, 1,489.9)(414.1, 1,482.3)(211.9, 594.0)
UC/C357.0555.0355.9
(178.4, 670.9)(272.7, 970.9)(240.7, 650.4)
SAP (mm Hg)134.0152.2145.2
(124.0, 149.6)(130.0, 170.0)(129.2, 158.2)
Urine specific gravity1.050b1.036c1.020a
(1.038, 1.060)(1.022, 1.052)(1.018, 1.028)
UC/UP2,469.9a2,732.5a,b2,009.2b
(1,401.7, 3,625.9)(1,594.5, 4,410.0)(1,582.8, 3,320.6)
UP/C0.16a0.17a0.15a
(0.13, 0.21)(0.10, 0.31)(0.11, 0.37)

Concentration ranges represent the stages of CKD outlined by the IRIS.9

Values with different superscript letters within a row are significantly (P <0.05) different from each other (1-way ANOVA, with Bonferroni correction).

Figure 1—
Figure 1—

Scatterplots of UC/C data for client-owned geriatric (≥ 9 years of age) cats classified according to their magnitude of proteinuria (nonproteinuric, UP/C < 0.2 [n = 114]; borderline proteinuric, UP/C = 0.2 to 0.4 [40]; proteinuric, UP/C > 0.4 [34]). Horizontal lines indicate median values. *Values differ significantly (P < 0.05) between indicated groups on ends of brackets.

Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.982

Cauxin as a predictor of azotemia—Clinical and cauxin data were compared (Figure 2; Table 2). A significant (P = 0.006) difference in logUC/C was evident in the nonazotemic cats at entry to the prospective cohort study between those cats that remained nonazotemic and those that developed azotemia by 12 months.

Figure 2—
Figure 2—

Scatterplots of UC/C data obtained at enrollment of client-owned geriatric (≥ 9 years of age) cats without (n = 19) and with (61) azotemia at their 12-month reexamination. See Figure 1 for key.

Citation: American Journal of Veterinary Research 71, 8; 10.2460/ajvr.71.8.982

Table 2—

Median (25th, 75th percentile) values for clinical variables and urinary cauxin in client-owned geriatric (≥ 9 years of age) cats before follow-up 12 months later to determine whether they were nonazotemic or azotemic.

VariableNonazotemic (n = 61)  
Cauxin (arbitrary units)696.0 (270.0, 1,693.9)836.0 (541.1, 1,342.4) 
UC/C324.0* (144.2, 667.4)660.6 (443.3, 1,003.7) 
SAP (mm Hg)134.0 (122, 153)153 (138, 165) 
Creatinine (mg/dL)1.51* (1.30, 1.71)1.81 (1.62,1.89) 
Urine specific gravity1.050* (1.033, 1.060)1.037 (1.022, 1.044) 
UP/C0.14* (0.10, 0.20)0.22 (0.15, 0.39) 

Values differ significantly (P < 0.05) between groups for indicated variable.

Discussion

The direct immunoassay developed in the study reported here provided a rapid and repeatable technique for the assessment of relative cauxin immunoreactivity in large numbers of clinical urine samples from geriatric cats. The major limitation to the assay results was the lack of a purified or synthetic cauxin to serve as a standard. Because of this, we were unable to quantify cauxin within urine but were able to examine patterns in data because the same pooled urine sample, generated from the urine of sexually intact male cats, was used as a standard throughout. Data were consequently reported as UC/C.

Urinary cauxin was either not detectable or generated a very low absorbance value in fresh urine samples and in urine samples that had been stored at 4°C. After urine samples had been stored at −20°C for 7 days, there was a significant increase in absorbance readings. However, prolonged storage at −20°C had no significant effect on relative cauxin concentrations. This suggested that the anti–cauxin peptide antibody used in our study may only recognize the denatured form of cauxin. Previously, this antibody had only been assessed via immunoblotting or immunohistochemical techniques in which urinary or tissue proteins are denatured.1,8 For future use of the assay reported here, all feline urine samples should be stored at −20°C for a minimum of 7 days.

Previous immunoblotting and immunohistochemical assays revealed that cauxin concentration and staining in renal proximal tubular cells declined with increasing azotemia and tubulointerstitial inflammation.8 However, the results of the present study indicated there was no significant difference in UC/C when cats were grouped according to their plasma creatinine concentration. This suggestred that even if precise cauxin quantification (ie, relative to a known mass of cauxin protein) had been possible, cauxin concentrations would not have differentiated among stages of azotemia.

On the other hand, there was a significant difference in UC/C when cats were grouped by magnitude of proteinuria, and correspondingly, there was a weak positive correlation between UC/C and UP/C values. If it is hypothesized that in cats with tubulointerstitial nephritis, proteinuria indicates damage at the level of the tubules and impaired tubular uptake mechanisms, then it can be speculated that increasing urine cauxin concentrations may also be the result of primary tubular cell damage. With end-stage renal disease, in which loss of tubules is extreme, cauxin concentrations may decrease substantially. This hypothesis was not investigated in the present study, but UC/UP values in our study decreased with increasing IRIS stage of creatinine.

Previous work has indicated that UP/C, urine albumin-to-creatinine concentration ratio, and plasma creatinine concentration are significantly higher and that urine specific gravity is significantly lower in cats that go on to develop azotemia within a 12-month period, compared with values in cats that remain nonazotaemic.10 Similarly, in the present study, on a population basis, UC/C was significantly higher in those cats that developed azotemia than in those remaining nonazotemic. However, as for all these potential markers, UC/C did not clearly differentiate between these groups of cats. Apart from the influence of cat age on the production of cauxin, little information is available regarding the mechanisms controlling the synthesis and secretion of cauxin. This would seem important information to determine whether cauxin may play a role as a marker of renal proximal tubular cell damage or whether, as the results of the present study would suggest, cauxin reflects just 1 component of the feline urinary proteome. It appears unlikely that UC/C alone will be predictive of the development of azotemia for individual cats in a clinical situation. Additional research is therefore warranted to evaluate UC/C within a multivariable model of biomarkers for the prediction of azotemia in cats.

ABBREVIATIONS

CKD

Chronic kidney disease

CV

Coefficient of variation

IRIS

International Renal Interest Society

logUC/C

Logarithmically transformed urine cauxin-to-creatinine concentration ratio

SAP

Systolic arterial blood pressure

TBSST

Tris-buffered saline solution plus 0.05% Tween 20

UC/C

Urine cauxin-to-creatinine concentration ratio

UC/UP

Urine cauxin-to-urine protein concentration ratio

UP/C

Urine protein-to-creatinine concentration ratio

a.

Parks Doppler model 811B, Parks Medical Electronics Inc, Las Vegas, Nev.

b.

Mistral 3000, Sanyo-Gallenkamp, Loughborough, Leicestershire, England.

c.

Idexx Laboratories, Wetherby, Yorkshire, England.

d.

HI 9224 pH meter, Hanna Instruments, Leighton Buzzard, Bedfordshire, England.

e.

Multistix Urine Chemistry Reagent Strips, Bayer Diagnostics, Newbury, Berkshire, England.

f.

Costar, Corning Inc, Corning, NY.

g.

Sigma-Aldrich, Poole, Dorset, England.

h.

P0448, DakoUK Ltd, Ely, Cambridgeshire, England.

i.

Tetramethylbenzidine liquid substrate, supersensitive, for ELISA (T4444), Sigma-Aldrich Co Ltd, Poole, Dorset, England.

j.

Spectronic Genesys 2, Patterson Scientific, Luton, Bedfordshire, England.

k.

Istin, Pfizer, Sandwich, Kent, England.

l.

GraphPad Prism, version 5.00 for Windows, GraphPad Software, San Diego, Calif.

m.

SPSS for Windows, version 15.0, SPSS Inc, Chicago, Ill.

References

  • 1.

    Miyazaki MKamiie KSoeta S, et al.Molecular cloning and characterization of a novel carboxylesterase-like protein that is physiologically present at high concentrations in the urine of domestic cats (Felis catus). Biochem J 2003;370:101110.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Holmes RSCox LAVandeberg JL. Mammalian carboxylesterase 5: comparative biochemistry and genomics. Comp Biochem Physiol Part D Genomics Proteomics 2008;3:195204.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Miyazaki MYamashita THosokawa M, et al.Species-, sex-, and age-dependent urinary excretion of cauxin, a mammalian carboxylesterase. Comp Biochem Physiol B Biochem Mol Biol 2006;145:270277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Miyazaki MYamashita TSuzuki Y, et al.A major urinary protein of the domestic cat regulates the production of felinine, a putative pheromone precursor. Chem Biol 2006;13:10711079.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Miyazaki MYamashita TTaira H, et al.The biological function of cauxin, a major urinary protein of the domestic cat (Felis catus). In: Hurst JLBeynon RJRoberts SC, et al, eds. Chemical signals in vertebrates 11. New York: Springer, 2008;5160.

    • Search Google Scholar
    • Export Citation
  • 6.

    Lulich JPOsborne CAO'Brien TD, et al.Feline renal failure: questions, answers, questions. Compend Contin Educ Pract Vet 1992;14:127152.

    • Search Google Scholar
    • Export Citation
  • 7.

    Ross SJOsborne CAKirk CA, et al.Clinical evaluation of dietary modification for treatment of spontaneous chronic kidney disease in cats. J Am Vet Med Assoc 2006;229:949957.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Miyazaki MSoeta SYamagishi N, et al.Tubulointerstitial nephritis causes decreased renal expression and urinary excretion of cauxin, a major urinary protein of the domestic cat. Res Vet Sci 2007;82:7679.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Elliott J. Staging chronic kidney disease. In: Elliott JGrauer GF, eds. BSAVA manual of canine and feline nephrology and urology. 2nd ed. Kundli Haryana, India: Replika Press Pvt Ltd, 2007;163.

    • Search Google Scholar
    • Export Citation
  • 10.

    Jepson REBrodbelt DVallance C, et al.Evaluation of predictors of the development of azotemia in cats. J Vet Intern Med 2009;23:806813.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Address correspondence to Dr. Jepson (rjepson@rvc.ac.uk).