The Veterinary Journal 198 (2013) 625–630
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Follow-up protein profiles in urine samples during the course of obstructive feline idiopathic cystitis G. Treutlein a, C.A. Deeg b, S.M. Hauck c, B. Amann b, K. Hartmann a, R. Dorsch a,⇑ a
Clinic of Small Animal Medicine, Center of Clinical Veterinary Medicine, LMU Munich, Germany Institute of Animal Physiology, Department of Veterinary Sciences, LMU Munich, Germany c Research Unit for Protein Science, Helmholtz Zentrum München, German Research Centre for Environmental Health, Neuherberg, Germany b
a r t i c l e
i n f o
Article history: Accepted 11 September 2013
Keywords: Cystitis Fibronectin Feline lower urinary tract disease Thioredoxin Urinary
a b s t r a c t Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder in cats, which often recurs. Published reports document increased urine fibronectin and thioredoxin concentrations in cats with FIC compared with healthy control cats. Therefore, these proteins might be of interest in the pathophysiology of FIC. The purpose of the present study was to evaluate variations in these urine proteins throughout the course of FIC by assessing their concentrations in urine specimens from cats with a history of obstructive FIC. Urine total protein (TP) was measured using the Bradford assay, while urine fibronectin and thioredoxin concentrations were determined by Western blot analysis. Urine TP was significantly higher in cats with obstructive FIC at presentation (day 0) than in healthy control cats (P < 0.01). There were significant decreases in urine TP in cats with obstructive FIC after 3 months (P < 0.01). Significantly higher urine fibronectin (P < 0.01) and thioredoxin (P < 0.05) concentrations were demonstrated in cats with FIC at day 0 compared to control cats, but there was no significant change over time (P > 0.05). Increased concentrations of these proteins over time might reflect ongoing structural and pathological alterations to functional processes in the urinary bladders of cats with obstructive FIC. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Disorders of the lower urinary tract are common in domestic cats, occurring in 1.3–1.7% of all cats examined at private veterinary practices (Lund et al., 1999). Cats with lower urinary tract disorders have similar clinical signs regardless of cause, and these are characterised by variable combinations of dysuria, stranguria, pollakiuria, haematuria and periuria (Gunn-Moore, 2003). The most common aetiologies for feline lower urinary tract disorders are feline idiopathic cystitis (FIC), followed by urolithiasis, bacterial urinary tract infection and urinary tract neoplasia (Lekcharoensuk et al., 2001; Gerber et al., 2005; Saevik et al., 2011). A common clinical sign, particularly in male cats with FIC, is obstruction of the lower urinary tract (Westropp et al., 2005). Urethritis, urethral muscle spasm and/or urethral plug formation have been proposed as causes of obstruction in FIC (Gerber et al., 2005; Westropp et al., 2005). To date, the aetiology of FIC remains obscure, although numerous hypotheses have been proposed over the last 30 years (Kruger and Osborne, 1993; Buffington et al., 1996; Lavelle et al., 2000; Buffington et al., 2002; Birder et al., 2005). FIC and human intersti-
⇑ Corresponding author. Tel.: +49 8921802650. E-mail address: [email protected] (R. Dorsch). 1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.09.015
tial cystitis (IC) share many clinical features, such as bladder pain, urgency, nocturia, the absence of an identifiable pathology, chronic signs, and the tendency to recur (Westropp and Buffington, 2002). The investigation of urine proteins has been applied to research investigating both human IC and FIC. Several potential urine biomarkers have been identified and examined for potential associations with disease severity and response to treatment (Keay et al., 2007; Liu et al., 2009; Goo et al., 2010). In veterinary medicine, only a small number of studies have investigated urine proteins in FIC. Recently, the widely expressed high molecular weight glycoprotein fibronectin was detected in the urine of cats with FIC, both with and without obstruction (Lemberger et al., 2011). Significantly higher urine fibronectin concentrations and lower fibronectin tissue signal intensities were demonstrated in cats with FIC compared to those without urinary tract disease and those with other urinary tract diseases, such as urolithiasis and bacterial urinary tract infection. Marked fibrosis of the bladder muscle layer and vascular walls, with the subsequent detachment and leakage of fibronectin into the urine, was suggested as the cause of increased urine fibronectin concentrations in FIC. Thioredoxin, a low molecular weight redox-regulating protein, was suggested as a novel potential interaction partner with fibronectin and co-precipitation with fibronectin was
626
G. Treutlein et al. / The Veterinary Journal 198 (2013) 625–630
demonstrated in an immunoprecipitation assay (Treutlein et al., 2012). Furthermore, cats with obstructive FIC had significantly higher urine thioredoxin concentrations than healthy control cats. This increase was thought to be due to apoptosis and oxidative stress, along with increased permeability of the urinary bladder wall. Therefore, both fibronectin and thioredoxin might play an important role in the pathophysiology of FIC. To the authors’ knowledge, there are no published studies documenting variations in urine protein concentrations throughout the course of FIC. The aim of the present study was to measure fibronectin and thioredoxin concentrations in urine samples from cats with obstructive FIC at different time points over a 6-month period and to evaluate whether changes in these proteins mirror the clinical picture. Materials and methods Specimens A total of 67 urine samples were collected from privately owned cats presented to the Clinic of Small Animal Medicine, Ludwig Maximilian University. The urine was obtained from two groups, namely, cats with FIC (n = 27 cats; n = 49 urine samples) and clinically healthy control cats (n = 18 cats; n = 18 urine samples). The inclusion criteria for the FIC group were clinical signs of lower urinary tract disease, such as haematuria, dysuria, pollakiuria and periuria, and obstruction of the lower urinary tract. Urinalysis including aerobic bacterial culture, abdominal radiography and ultrasound, were performed on all cats in the FIC group. Cats were excluded if marked crystalluria was present or if there was evidence of urolithiasis, mineralised urethral plug formation, bacterial urinary tract infection, or neoplasia. Cats were also excluded if the aerobic urine culture revealed bacterial growth. The maximum duration of clinical signs before presentation was 48 h. None of the cats from which follow-up specimens were collected had experienced prior episodes of obstructive or non-obstructive FLUTD. All cats in the FIC group were treated symptomatically for obstructive feline lower urinary tract disease (FLUTD) with fluid therapy, using buffered fluids adjusted to the hydration status of the cat and to the presence of post-obstructive diuresis, with analgesia and placement of an indwelling urinary catheter for 48 h. Phenoxybenzamine was administered as soon as the cats were stable. The healthy control group consisted of cats that were presented to the same clinic for routine health care examinations. Cats were included if they had no history of prior urinary tract disease, no clinical signs of urinary tract disease, and results within the reference range on a complete urinalysis, including specific gravity (USG), dipstick and sediment. All procedures performed for the study were medically indicated. Urinalysis in the healthy cat group was part of a wellness programme. Informed owner consent was obtained for all procedures and for the use of the specimens excess to those required for diagnostic purposes. Urinalysis and total protein (TP) quantification All urine samples were collected by cystocentesis. Day 0 urine specimens were collected from cats in the FIC group at initial presentation, prior to treatment. Follow-up urine samples (n = 22) were collected from seven cats at time points which included 2 weeks, 1 month, 3 months and 6 months after day 0. Complete urinalysis (USG, dipstick and urine sediment) was performed within 30 min after sampling.
A refractometer (Atago) was used to determine USG. Urine was then analysed with reagent strips (Combur-9; Roche Diagnostics). After centrifugation at 2000 g for 5 min, the urine sediment was examined microscopically. Aliquots were prepared from the supernatants and stored at 80 °C until further processing. Urine TP in the supernatant was determined by the Bradford assay (Sigma–Aldrich). Gel electrophoresis, Western blot analysis and signal quantification Equal amounts of TP (5 lg) were separated from urine supernatants using sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and were blotted semi-dry onto polyvinyldifluoride membranes (GE Healthcare). Non-specific binding was blocked with 1% polyvinylpyrrolidone in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) for 1 h at room temperature (RT). The blots were incubated with the primary antibody at 4 °C overnight. Rabbit anti-human fibronectin antibody (Thermo Fisher, 1:1000), and rabbit anti-human thioredoxin antibody (Abcam, 1:400) were used to detect feline fibronectin and thioredoxin, respectively. The blots were washed three times in PBS-T and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 1 h at RT (Serotec, 1:5000). After 12 further washing steps in PBS-T, the signals were detected by enhanced chemiluminescence on X-ray films (Euromed; Christiansen). Finally, the films were scanned with a transmission scanner and signals were quantified by densitometry using ImageQuantTL software v2005 (GE Healthcare). Statistical analysis Prism 5.04 software (GraphPad) was used for the statistical analysis. Kolmogorov–Smirnov tests were performed to evaluate the data distribution (USG, protein, fibronectin and thioredoxin) in each group at different time points. Kruskal–Wallis test and Dunn’s multiple comparison tests were used to evaluate differences in continuous data (USG, protein, fibronectin and thioredoxin) among cats with FIC at days 0 and 14, and after 1, 3 and 6 months, and for comparison between groups. An intention-to-treat analysis (ITT) was performed, with the last value carried forward for TP, fibronectin and thioredoxin data from follow-up urine specimens in the FIC group. Friedman’s and Dunn’s multiple comparison tests were used to compare data between time points. Chi-squared tests were used to compare erythrocytes per high power field (hpf) and leukocytes/hpf. Data were categorised for erythrocytes/hpf (1, 100) and leukocytes/hpf (1, 0–4; 2, >5). Statistical significance was set at P < 0.05.
Results Animals Cats in the FIC group (n = 27) had a mean age of 5.3 ± 2.2 years (median 4.5; range 2–10 years); all were castrated males. Twentysix cats were European shorthairs and one was a Maine Coon. Nineteen cats in the FIC group presented with a first episode of FIC, four experienced a second episode, three had a third episode and one cat had experienced more than three episodes. All seven cats included in the follow-up sample study were initially presented at their first episode of obstructive or non-obstructive FLUTD. Five cats with FIC showed no clinical lower urinary tract signs on follow-up examinations. However, there was stranguria and
Table 1 Urinalysis results and signal intensities for fibronectin and thioredoxin in cats with obstructive feline idiopathic cystitis (FIC) at day of initial presentation (day 0) and in healthy control cats. Cats with obstructive FIC
USG (g/g) a Erythrocytes/hpf b Leukocytes/hpf c Protein (mg/mL) d Fibronectin signal intensity e Thioredoxin signal intensity f
Healthy control cats
Mean ± SD
Median (range)
Mean ± SD
Median (range)
1.039 ± 0.011
1.042 (1.017–1.060) >100 (100) >5 (0–4 to >12) 1.7 (0.21–13.9) 25167 (12122–121543) 69818 (24288–197268)
1.051 ± 0.010
1.055 (1.035–1.060)
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Follow-up protein profiles in urine samples during the course of obstructive feline idiopathic cystitis G. Treutlein a, C.A. Deeg b, S.M. Hauck c, B. Amann b, K. Hartmann a, R. Dorsch a,⇑ a
Clinic of Small Animal Medicine, Center of Clinical Veterinary Medicine, LMU Munich, Germany Institute of Animal Physiology, Department of Veterinary Sciences, LMU Munich, Germany c Research Unit for Protein Science, Helmholtz Zentrum München, German Research Centre for Environmental Health, Neuherberg, Germany b
a r t i c l e
i n f o
Article history: Accepted 11 September 2013
Keywords: Cystitis Fibronectin Feline lower urinary tract disease Thioredoxin Urinary
a b s t r a c t Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder in cats, which often recurs. Published reports document increased urine fibronectin and thioredoxin concentrations in cats with FIC compared with healthy control cats. Therefore, these proteins might be of interest in the pathophysiology of FIC. The purpose of the present study was to evaluate variations in these urine proteins throughout the course of FIC by assessing their concentrations in urine specimens from cats with a history of obstructive FIC. Urine total protein (TP) was measured using the Bradford assay, while urine fibronectin and thioredoxin concentrations were determined by Western blot analysis. Urine TP was significantly higher in cats with obstructive FIC at presentation (day 0) than in healthy control cats (P < 0.01). There were significant decreases in urine TP in cats with obstructive FIC after 3 months (P < 0.01). Significantly higher urine fibronectin (P < 0.01) and thioredoxin (P < 0.05) concentrations were demonstrated in cats with FIC at day 0 compared to control cats, but there was no significant change over time (P > 0.05). Increased concentrations of these proteins over time might reflect ongoing structural and pathological alterations to functional processes in the urinary bladders of cats with obstructive FIC. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Disorders of the lower urinary tract are common in domestic cats, occurring in 1.3–1.7% of all cats examined at private veterinary practices (Lund et al., 1999). Cats with lower urinary tract disorders have similar clinical signs regardless of cause, and these are characterised by variable combinations of dysuria, stranguria, pollakiuria, haematuria and periuria (Gunn-Moore, 2003). The most common aetiologies for feline lower urinary tract disorders are feline idiopathic cystitis (FIC), followed by urolithiasis, bacterial urinary tract infection and urinary tract neoplasia (Lekcharoensuk et al., 2001; Gerber et al., 2005; Saevik et al., 2011). A common clinical sign, particularly in male cats with FIC, is obstruction of the lower urinary tract (Westropp et al., 2005). Urethritis, urethral muscle spasm and/or urethral plug formation have been proposed as causes of obstruction in FIC (Gerber et al., 2005; Westropp et al., 2005). To date, the aetiology of FIC remains obscure, although numerous hypotheses have been proposed over the last 30 years (Kruger and Osborne, 1993; Buffington et al., 1996; Lavelle et al., 2000; Buffington et al., 2002; Birder et al., 2005). FIC and human intersti-
⇑ Corresponding author. Tel.: +49 8921802650. E-mail address: [email protected] (R. Dorsch). 1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2013.09.015
tial cystitis (IC) share many clinical features, such as bladder pain, urgency, nocturia, the absence of an identifiable pathology, chronic signs, and the tendency to recur (Westropp and Buffington, 2002). The investigation of urine proteins has been applied to research investigating both human IC and FIC. Several potential urine biomarkers have been identified and examined for potential associations with disease severity and response to treatment (Keay et al., 2007; Liu et al., 2009; Goo et al., 2010). In veterinary medicine, only a small number of studies have investigated urine proteins in FIC. Recently, the widely expressed high molecular weight glycoprotein fibronectin was detected in the urine of cats with FIC, both with and without obstruction (Lemberger et al., 2011). Significantly higher urine fibronectin concentrations and lower fibronectin tissue signal intensities were demonstrated in cats with FIC compared to those without urinary tract disease and those with other urinary tract diseases, such as urolithiasis and bacterial urinary tract infection. Marked fibrosis of the bladder muscle layer and vascular walls, with the subsequent detachment and leakage of fibronectin into the urine, was suggested as the cause of increased urine fibronectin concentrations in FIC. Thioredoxin, a low molecular weight redox-regulating protein, was suggested as a novel potential interaction partner with fibronectin and co-precipitation with fibronectin was
626
G. Treutlein et al. / The Veterinary Journal 198 (2013) 625–630
demonstrated in an immunoprecipitation assay (Treutlein et al., 2012). Furthermore, cats with obstructive FIC had significantly higher urine thioredoxin concentrations than healthy control cats. This increase was thought to be due to apoptosis and oxidative stress, along with increased permeability of the urinary bladder wall. Therefore, both fibronectin and thioredoxin might play an important role in the pathophysiology of FIC. To the authors’ knowledge, there are no published studies documenting variations in urine protein concentrations throughout the course of FIC. The aim of the present study was to measure fibronectin and thioredoxin concentrations in urine samples from cats with obstructive FIC at different time points over a 6-month period and to evaluate whether changes in these proteins mirror the clinical picture. Materials and methods Specimens A total of 67 urine samples were collected from privately owned cats presented to the Clinic of Small Animal Medicine, Ludwig Maximilian University. The urine was obtained from two groups, namely, cats with FIC (n = 27 cats; n = 49 urine samples) and clinically healthy control cats (n = 18 cats; n = 18 urine samples). The inclusion criteria for the FIC group were clinical signs of lower urinary tract disease, such as haematuria, dysuria, pollakiuria and periuria, and obstruction of the lower urinary tract. Urinalysis including aerobic bacterial culture, abdominal radiography and ultrasound, were performed on all cats in the FIC group. Cats were excluded if marked crystalluria was present or if there was evidence of urolithiasis, mineralised urethral plug formation, bacterial urinary tract infection, or neoplasia. Cats were also excluded if the aerobic urine culture revealed bacterial growth. The maximum duration of clinical signs before presentation was 48 h. None of the cats from which follow-up specimens were collected had experienced prior episodes of obstructive or non-obstructive FLUTD. All cats in the FIC group were treated symptomatically for obstructive feline lower urinary tract disease (FLUTD) with fluid therapy, using buffered fluids adjusted to the hydration status of the cat and to the presence of post-obstructive diuresis, with analgesia and placement of an indwelling urinary catheter for 48 h. Phenoxybenzamine was administered as soon as the cats were stable. The healthy control group consisted of cats that were presented to the same clinic for routine health care examinations. Cats were included if they had no history of prior urinary tract disease, no clinical signs of urinary tract disease, and results within the reference range on a complete urinalysis, including specific gravity (USG), dipstick and sediment. All procedures performed for the study were medically indicated. Urinalysis in the healthy cat group was part of a wellness programme. Informed owner consent was obtained for all procedures and for the use of the specimens excess to those required for diagnostic purposes. Urinalysis and total protein (TP) quantification All urine samples were collected by cystocentesis. Day 0 urine specimens were collected from cats in the FIC group at initial presentation, prior to treatment. Follow-up urine samples (n = 22) were collected from seven cats at time points which included 2 weeks, 1 month, 3 months and 6 months after day 0. Complete urinalysis (USG, dipstick and urine sediment) was performed within 30 min after sampling.
A refractometer (Atago) was used to determine USG. Urine was then analysed with reagent strips (Combur-9; Roche Diagnostics). After centrifugation at 2000 g for 5 min, the urine sediment was examined microscopically. Aliquots were prepared from the supernatants and stored at 80 °C until further processing. Urine TP in the supernatant was determined by the Bradford assay (Sigma–Aldrich). Gel electrophoresis, Western blot analysis and signal quantification Equal amounts of TP (5 lg) were separated from urine supernatants using sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and were blotted semi-dry onto polyvinyldifluoride membranes (GE Healthcare). Non-specific binding was blocked with 1% polyvinylpyrrolidone in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) for 1 h at room temperature (RT). The blots were incubated with the primary antibody at 4 °C overnight. Rabbit anti-human fibronectin antibody (Thermo Fisher, 1:1000), and rabbit anti-human thioredoxin antibody (Abcam, 1:400) were used to detect feline fibronectin and thioredoxin, respectively. The blots were washed three times in PBS-T and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 1 h at RT (Serotec, 1:5000). After 12 further washing steps in PBS-T, the signals were detected by enhanced chemiluminescence on X-ray films (Euromed; Christiansen). Finally, the films were scanned with a transmission scanner and signals were quantified by densitometry using ImageQuantTL software v2005 (GE Healthcare). Statistical analysis Prism 5.04 software (GraphPad) was used for the statistical analysis. Kolmogorov–Smirnov tests were performed to evaluate the data distribution (USG, protein, fibronectin and thioredoxin) in each group at different time points. Kruskal–Wallis test and Dunn’s multiple comparison tests were used to evaluate differences in continuous data (USG, protein, fibronectin and thioredoxin) among cats with FIC at days 0 and 14, and after 1, 3 and 6 months, and for comparison between groups. An intention-to-treat analysis (ITT) was performed, with the last value carried forward for TP, fibronectin and thioredoxin data from follow-up urine specimens in the FIC group. Friedman’s and Dunn’s multiple comparison tests were used to compare data between time points. Chi-squared tests were used to compare erythrocytes per high power field (hpf) and leukocytes/hpf. Data were categorised for erythrocytes/hpf (1, 100) and leukocytes/hpf (1, 0–4; 2, >5). Statistical significance was set at P < 0.05.
Results Animals Cats in the FIC group (n = 27) had a mean age of 5.3 ± 2.2 years (median 4.5; range 2–10 years); all were castrated males. Twentysix cats were European shorthairs and one was a Maine Coon. Nineteen cats in the FIC group presented with a first episode of FIC, four experienced a second episode, three had a third episode and one cat had experienced more than three episodes. All seven cats included in the follow-up sample study were initially presented at their first episode of obstructive or non-obstructive FLUTD. Five cats with FIC showed no clinical lower urinary tract signs on follow-up examinations. However, there was stranguria and
Table 1 Urinalysis results and signal intensities for fibronectin and thioredoxin in cats with obstructive feline idiopathic cystitis (FIC) at day of initial presentation (day 0) and in healthy control cats. Cats with obstructive FIC
USG (g/g) a Erythrocytes/hpf b Leukocytes/hpf c Protein (mg/mL) d Fibronectin signal intensity e Thioredoxin signal intensity f
Healthy control cats
Mean ± SD
Median (range)
Mean ± SD
Median (range)
1.039 ± 0.011
1.042 (1.017–1.060) >100 (100) >5 (0–4 to >12) 1.7 (0.21–13.9) 25167 (12122–121543) 69818 (24288–197268)
1.051 ± 0.010
1.055 (1.035–1.060)
