Lower urinary tract disease is common in domestic cats, affecting 1.5% of cats examined at private veterinary practices.1 It is often a cause of surrender of cats to animal shelters or even euthanasia.2 In cats, the most frequent cause of lower urinary tract disease is IdC, accounting for 55% to 69% of cases.3–5 Various hypotheses regarding the etiopathogenesis of IdC have been proposed, but research over the last 30 years has failed to find a consistent cause.5,6 Cats with any disease of the lower urinary tract have similar clinical signs, regardless of the cause.5 Therefore, to make a diagnosis of IdC, other disease processes (eg, UTI, urolithiasis, and neoplasia) need to be excluded by use of several diagnostic means.
Feline IdC shares common features with ItC in humans.7 Interstitial cystitis results in clinical signs similar to those that develop in cats with IdC, and it is also a chronic disease with a tendency to recur.8 Histologic examination of the urinary bladder wall in humans with ItC and cats with IdC reveals similar pathological changes, including petechial hemorrhages, denudation, tears, thinning of the transitional cell epithelium, and, depending on the form of disease (ie, ulcerative vs non-ulcerative), the presence of ulcers extending into the lamina propria.9 The consensus in veterinary as well as human medicine is that IdC and ItC are multifactorial disease syndromes involving the urinary bladder.7,8,10–13
With regard to IdC, known contributing factors are environmental influences and changes in the autonomic nervous system, which generate an increased stress response of affected cats.6,14,15 One recent hypothesis is that epigenetic modulation of gene expression caused by adverse experiences in early life sensitizes affected cats to increased stress response, long-term neuroendocrine abnormalities, and eventually clinical disease.16 In human medicine, 3 interesting urine biomarkers—an antiproliferative factor, a heparin-binding epidermal growth factor-like growth factor and an epidermal growth factor—and their association with ItC were discovered in 1996.17 Antiproliferative factor inhibits normal urinary bladder epithelial growth in vitro17,18 and is upregulated in patients with ItC.17–19 Heparin-binding epidermal growth factor–like growth factor is important for replication of epithelial cells,19 and it is downregulated by antiproliferative factor in patients with ItC. Epidermal growth factor stimulates epithelial cell proliferation, and in patients with ItC, it is upregulated by antiproliferative factor. Assessments of these 3 biomarkers have been shown to clearly distinguish between patients with ItC and healthy controls, but such testing is not available for use in human clinical practice yet and still needs to be validated in independent laboratories.20 To our knowledge, urine biomarkers for IdC in cats have not been investigated. The purpose of the study reported here was to characterize and compare the urine protein content in healthy cats and cats with IdC, UTI, or urolithiasis. We speculated that the detection of specific proteins in the urine of cats with IdC would provide information about the possible etiopathogenesis of IdC and identify potential urine biomarkers for this disease in cats.
Materials and Methods
Animals—The purpose of the study was to characterize and compare the urine protein content in control cats and cats with IdC, UTI, or urolithiasis. Involved cats were privately owned and had no urinary tract disease or naturally occurring urinary tract disease. These cats were evaluated at the Clinic of Small Animal Medicine, Ludwig Maximilian University, Munich, from January 2008 through March 2009. Every cat evaluated at the Clinic of Small Animal Medicine in January 2008 through March 2009 that met the inclusion criteria for 1 of the 4 groups (ie, cats with IdC, UTI, or urolithiasis and cats with no apparent urinary tract disease or abnormalities [controls]) was included in the study. All procedures performed in any of the cats involved in the study were medically indicated. Informed owner consent was obtained regarding procedures and the use of unused samples (including body parts) for research purposes. Inclusion criteria for cats with IdC were micturition problems, such as hematuria, dysuria, pollakiuria, and periuria. To determine eligibility for inclusion in this study group, ultrasonography and radiography of the urinary tract, urinalysis, and bacterial culture of a urine sample were performed. Cats were excluded if they had crystalluria, bacteriuria, urolithiasis, or evidence of urinary tract neoplasia, or if results of bacterial culture of a urine sample were positive. In cats that had previous episodes of lower urinary tract disease, these episodes had been attributed to IdC on the basis of results of similar diagnostic testing procedures at the Clinic of Small Animal Medicine. Cats with IdC that had or did not have urethral obstruction were included in the study.
Control cats were those treated for diseases that did not involve the urinary tract or evaluated for health examinations and vaccinations at the Clinic of Small Animal Medicine. Inclusion criteria for the control cats were normal physiologic micturition, an absence of any history of micturition abnormalities, and detection of no abnormalities via urinalysis on the day of inclusion. Furthermore, ultrasonography of the entire urinary tract as well as measurement of serum creatinine and BUN concentrations was performed in these cats to confirm that that each had a healthy urinary tract.
Inclusion criteria for cats with UTI were micturition problems and positive results of bacterial culture of their urine. Furthermore, cats were confirmed not to have urolithiasis on the basis of results of ultrasonography and, in most cases, radiography. For inclusion in the urolithiasis group, the presence of a urolith was confirmed via ultrasonography or radiography.
From January 2008 through March 2009, 110 cats were evaluated at the Clinic of Small Animal Medicine for lower urinary tract disease. Among these cats, 18 cats with IdC, 18 cats with no apparent urinary tract disease or abnormalities (controls), 12 cats with UTI, and 12 cats with urolithiasis met the inclusion criteria for the respective study group and were included in the study. Ten cats with IdC as well as 5 cats with UTI and 9 cats with urolithiasis had urethral obstruction. Of the 5 obstructed cats with UTI, 2 were completely obstructed and 3 were partially obstructed.
The portion of the study in which urinary bladder biopsy specimens underwent histologic examination and immunohistochemical analysis for fibronectin involved 6 additional cats (2 with obstructive IdC and 4 controls). The 4 control cats had been submitted for postmortem examination to the Clinic of Pathology of the Ludwig Maximilian University, Munich. These cats had hepatic lipidosis, feline panleukopenia, a brain tumor, or congestive heart failure. For the 2 cats with obstructive IdC, the owners declined treatment and elected euthanasia of their pet.
Sample collection—Urine samples were collected from 18 cats with no apparent urinary tract disease or abnormalities (controls), 18 cats with IdC, 12 cats with UTI, and 12 cats with urolithiasis for use in the study. Urine was obtained by means of cystocentesis in 7 cats with nonobstructive IdC, in 4 cats with obstructive IdC, in 9 cats with UTI (including the 5 cats with obstructive UTI), and in 5 cats with urolithiasis (including 2 cats with obstruction), as well as in all 18 controls. Catheterization was performed in 1 cat with nonobstructive IdC and in 6 cats with obstructive IdC. Moreover, urine samples were obtained via catheterization from 7 cats in the urolithiasis group (all of these cats had urethral obstruction) and from 3 cats in the UTI group (all of which were obstructed). In cats with IdC, UTI, or urolithiasis, the urine sample was obtained within a maximum of 24 hours after the onset of clinical signs. Urine was centrifuged for 5 minutes within 30 minutes after urine collection, sediments were examined immediately, and supernatants were divided into aliquots and stored at −80°C on the day of sample collection until further processing.
In 6 additional cats (2 with obstructive IdC and 4 controls), full-thickness urinary bladder biopsies were obtained. To obtain biopsy specimens, urinary bladders were excised in their entirety, and sections of various areas were prepared. Bladders were excised within 30 minutes after euthanasia. Biopsy specimens were fixed in Bouin solutiona and embedded in paraffin.b
Urinalysis—A refractometerc was used to determine urine specific gravity. Urine composition was analyzed with reagent strips,d and unstained sediments, which were obtained by centrifugation within 30 minutes after collection, were examined microscopically.
Protein quantification and 1-D gel electrophoresis—Protein content in each urine sample supernatant obtained from cats in the 4 groups was quantified by use of a Bradford assay.e One-dimensional gels were loaded with urine sample supernatants so that each well contained 5 μg of protein. One-dimensional gel electrophoresis was performed on samples from all cats in the 4 groups. Supernatants were separated by a 10% gradient SDS-PAGE,f and 1-D gels were subsequently stained with Coomassie blue.g
MS—From 1-D electrophoresis gels of urine obtained from a representative cat with IdC, bands were excised, destained, processed by proteolysis with trypsin,21,22 and analyzed by use of MALDI-TOF–TOF pep-tide mass fingerprinting and MS–MS with a tandem mass spectrometer.h A nonredundant mammalian database was used for candidate search. Combined pep-tide mass fingerprinting and MS–MS queries were performed by use of a database search enginei embedded into a comprehensive data analysis packagej on a curated protein sequence databasek (270, 778 sequences; 99, 412, 397 residues) and a metadatabasel (3, 239, 079 sequences; 1, 079, 594, 700 residues) for mammalian proteins, as described before by Lemberger et al.23 Additionally, a search was performed for bacterial proteins by use of a eubacteria database of the curated protein sequence database (131, 025 sequences). A protein was regarded as identified if the probability-based molecular weight search score based on an algorithmm was significant (P < 0.05) for the respective database (protein scores > 60 were significant for mammalian proteins, scores > 64 were significant for eubacteria in the curated protein sequence database, and scores > 68 were significant for the metadatabase [mammalian]), if the matched peptide masses were abundant in the spectrum, and if the theoretical masses of the significant hit fit the experimentally observed values. To date, there are 3, 053 entries for Felis catus in the curated protein sequence database.
Western blotting, image analysis, and protein quantification—One-dimensional gels for western blots were loaded with urine samples so that each well contained 5 μg of protein. Western blotting was performed by use of nitrocellulose membranes.n Unspecific binding was blocked with 1% polyvinylpyrrolidone in PBS solution and Tween-20 for 1 hour. Blots were subsequently incubated with primary antibody (polyclonal rabbit anti–fibronectin antibodyo [1:1, 000]) overnight at 4°C. Afterward, blots were washed and incubated with horseradish peroxidase–conjugated secondary antibody (polyclonal anti-rabbit antibodyp [1:3, 000]). Signals were detected with enhanced chemiluminescence on radiographic films. Quantification of western blot signals was performed with general image analysis softwareq after scanning the films on a transmission scannerr by use of associated software, s as described by Lemberger et al.23
Histologic examination and immunohistochemical analysis of urinary bladder tissue—Sections of urinary bladder biopsy specimens obtained from control cats and cats with obstructive IdC were stained with Masson-Goldner stain for assessment of histopathologic changes associate with IdC. Fibronectin expression patterns in bladder tissues from control cats and bladder tissues from cats with IdC were evaluated immunohistochemically by use of the same antibody as used in western blotting procedures. Antigen retrieval was performed at 99°C for 15 minutes in 0.1M EDTA-NaOH buffer (pH, 8.8). Anti-rabbit IgG coupled to fluorescent dyet was used for visualization of ligated fibronectin antibody. A fluorescent stainu was used to stain cell nuclei. Tissue sections were examined microscopically and photographed with a microscope and camera system.v Observation of fluorescence in tissue sections was performed with microscope software.w
Statistical analysis—Statistical analyses were performed by use of a data analysis package.x Data are reported as mean ± SD and median and range. A Kolmogorov-Smirnov test was used to check for data distribution. A t test was applied to compare normally distributed data (protein concentrations) from cats in the control group with data from cats with IdC, UTI, or urolithiasis. For comparison of western blot signal intensities between the control group and cats with IdC, UTI, or urolithiasis, a Mann-Whitney U test was used. Comparisons of fibronectin in obstructed and nonob-structed cats with IdC and comparisons of albumin and fibronectin signal intensities in cats with IdC were also performed by use of a Mann-Whitney U test. Values of P ≤ 0.05 were considered significant.
Results
Animals—Urine samples were collected from control cats (n = 18) and cats with IC (18), UTI (12), and urolithiasis (12). Postmortem urinary bladder biopsy specimens were obtained from another 2 cats with obstructive IC and 4 control cats.
Among the cats from which urine samples were collected, mean ± SD age of the cats with IdC was 6.2 + 3.4 years (median, 6.5 years; range, 2 to 14 years). In the UTI group, mean age was 10.9 ± 4.1 years (median, 10 years; range, 3 to 18 years); in the urolithiasis group, mean age was 7.3 ± 2.9 years (median, 7 years; range, 3 to 12 years). Mean age of the control cats was 9.2 ± 6.6 years (median, 10 years; range, 1 to 18 years).
In the IdC, control, UTI, and urolithiasis groups, there were 14, 10, 7, and 10 castrated males, respectively; 2, 2, 0, and 1 sexually intact males, respectively; and 2, 3, 5, and 1 spayed females, respectively. The control group also included 3 sexually intact females. In the IdC, control, UTI, and urolithiasis groups, there were 14, 11, 11, and 8 European Shorthairs, respectively. In the IdC group, there also was 1 Norwegian Forest Cat, 2 Siamese, and 1 British Shorthair. In the control group, there were 3 Maine Coons, 3 Persians, and 1 Siamese. In the UTI group, there was 1 Persian, and in the urolithiasis group, there were 2 Persians, 1 Russian Blue, and 1 British Shorthair.
Ten cats with IdC as well as 5 cats with UTI and 9 cats with urolithiasis had urethral obstruction. Of the 5 obstructed cats with UTI, 2 were completely obstructed and 3 were partially obstructed. Obstructed cats had unsuccessful or minimally successful voiding attempts; on palpation, the urinary bladder was enlarged and signs of pain were elicited. Palpation of the bladder did result in release of some drops of urine in partially obstructed cats but no urine stream. Of the 18 cats in the IdC group, 11 were undergoing their first episode of IdC, 4 were undergoing their second episode, and 2 were undergoing their third episode; 1 cat had had recurring episodes during the preceding 3 years. Most cats (n = 14) had moderate clinical signs; only 4 cats had severe clinical signs such as signs of depression, severe dehydration, and bradycardia. These clinical signs were attributed to severe postrenal azotemia, metabolic acidosis, and hyperkalemia.
In the UTI group, all cats confirmed with UTI for the first time had moderate clinical signs. With the exception of 1 cat in which uroliths were diagnosed 2 years earlier, cats with urolithiasis were identified with this disease for the first time. Two of the 9 cats with urolithiasis and urethral obstruction had severe clinical signs (similar to those described for severely affected cats with IdC); the other 8 cats with urolithiasis and urethral obstruction had moderate clinical signs. The 3 cats with nonobstructive urolithiasis had mild clinical signs.
The mean ± SD age of the control cats from which bladder biopsy specimens were obtained was 3.45 ± 2.6 years. Two cats were castrated males, 1 was a sexually intact female, and 1 was a sexually intact male; all 4 were European Shorthair cats. The 2 cats with IdC from which bladder biopsy specimens were obtained were 5- and 8-year-old European Shorthairs, both castrated males. Both cats had their first episode of IdC; at the time of evaluation at the Clinic of Small Animal Medicine, each cat had urethral obstruction of < 24 hours' duration and moderate clinical signs.
Urinalysis results—In cats with IdC, median urine specific gravity was 1.042 (range, 1.027 to > 1.050). All 18 cats had macroscopic and microscopic hematuria; in urine sediments of all cats, erythrocytes were too numerous to count (> 100 erythrocytes/hpf). Seven cats had > 12 leukocytes/hpf, and 11 cats had 0 to 4 leukocytes/hpf. No other urine sediment abnormalities were detected. Reagent strip testing of urine samples revealed 4+ blood in all 18 cats, 1+ protein in 2 cats, 2+ protein in 5 cats, and 3+ protein in 11 cats. One cat had 1+ bilirubin. Glucose, urobilinogen, or ketones were not detected in any of the cats' urine samples. Urine pH was 6 in 12 cats, 7 in 3 cats, and 8 in 3 cats. In control cats, median urine specific gravity was 1.040 (range, 1.025 to 1.050). Except for mild microscopic hematuria and mild microscopic proteinuria (0 to 20 erythrocytes/hpf) in 8 control cats, which was attributed to cystocentesis, results of urine sediment examinations were unremarkable. Reagent strip testing of urine samples revealed no blood in 7 cats and 1+ blood in 11 cats; 1+ protein was detected in the latter 11 cats. No bilirubin, urobilinogen, or ketones were detected in any of the cats' urine samples. Urine pH was 5 in 3 cats, 6 in 13 cats, and 7 in 2 cats. In cats with UTI, median urine specific gravity was 1.024 (range, 1.014 to 1.040). All 12 cats with UTI had macroscopic and microscopic hematuria; in their urine sediments, erythrocytes were too numerous to count (> 100 erythrocytes/hpf) for 9 cats, and there were 50 to 100 erythrocytes/hpf for 3 cats. Urine sediment examinations revealed > 12 leukocytes/hpf in 5 cats, 5 to 12 leukocytes/hpf in 2 cats, and 0 to 4 leukocytes/hpf in 5 cats. In cats with UTI, reagent strip testing of urine samples revealed 2+ blood in 1 cat, 3+ blood in 2 cats, and 3+ blood in 9 cats. Moreover, reagent strip testing revealed 1+ protein in 3 cats, 2+ protein in 5 cats, and 3+ protein in 5 cats. In their urine samples, 3 cats had 2+ glucose and 1 cat had 1+ glucose; 1 cat also had 1+ bilirubin and 1+ urobilinogen. No ketones were detected in any of the cats' urine samples. Urine pH was 5 in 2 cats, 6 in 5 cats, 7 in 4 cats, and 8 in 1 cat. In cats with urolithiasis, median urine specific gravity was 1.037 (range, 1.015 to 1.045). Ten cats with urolithiasis had macroscopic and microscopic hematuria, and urine sediments contained > 100 erythrocytes/hpf. In 2 cats, there was no macroscopic hematuria, and the urine sediments contained 0 to 8 erythrocytes/hpf. Urine sediment examinations revealed were > 12 leukocytes/hpf in 5 cats, 5 to 12 leukocytes/hpf in 2 cats, and 0 to 4 leukocytes/hpf in 5 cats. In cats with urolithiasis, reagent strip testing of urine samples revealed that 2 cats had 2+ blood, 1 cat had 3+ blood, and 9 cats had 4+ blood. Two cats had 1+ protein, 4 cats had 2+ protein, and 6 cats had 3+ protein in their urine samples. Glucose, bilirubin, urobilinogen, or ketones were not detected in any of the cats with urolithiasis. Urine pH was 5 in 2 cats, 6 in 4 cats, 7 in 4 cats, and 8 in 2 cats.
Urine protein quantification—Compared to the protein content in urine samples obtained from control cats (mean concentration, 0.81 ± 0.24 mg/mL), protein contents in the urine samples from cats with IdC (mean concentration, 1.85 ± 0.49 mg/mL), UTI (mean concentration, 3.44 ± 4.14 mg/mL), and urolithiasis (mean concentration, 5.12 ± 4.45 mg/mL) were significantly (P < 0.001) higher. The differences in urine protein content between cats with UTI and cats with IdC (P = 0.13) and between cats with UTI and cats with urolithiasis (P = 0.32) were not significant. There was a significant (P = 0.006) difference in urine protein content between cats with urolithiasis and cats with IdC; cats with urolithiasis had significantly higher urine protein concentration.
1-D gel electrophoresis—Macroscopic comparison of urine protein bands in 1-D electrophoresis gels (Figure 1) revealed different expression patterns between the IdC group and the control group. Cats with IdC had 13 urine protein bands, and the expression pattern within the group was nearly uniform; only band 13 was absent in gels for 2 cats with IdC, and it was less intense in gels for 6 cats. Four urine protein bands (bands 2, 3, 12, and 13) expressed in gels for cats with IdC were completely absent in gels for the control cats. Band 6 was comparatively less intense in controls. In addition, cats in the control group also had quite uniform expression patterns, with 10 urine protein bands. All 13 urine protein bands in a gel for 1 representative cat with IdC were excised and subsequently analyzed by means of tandem MS.
Results of 1-D gel electrophoresis (A and C) and western plot strips (B and D) of proteins in urine samples collected from cats with IdC (A and B) and cats with no urinary tract disease or abnormalities (controls; C and D). The 1-D gel electrophoresis gels for urine samples from a representative cat with IdC and a representative control cat (A and C, respectively) and the western blot strips for urine samples from the same representative cat with IdC and the same representative control cat (B and D, respectively) are illustrated. The 1-D gels were stained with Coomassie blue. Proteins bands were analyzed, and 13 proteins (1 to 13) were identified by means of MALDI-TOF–TOF. The left bar represents the marker (prestained protein laddery). Band number 1 was identified as fibronectin. The positioning of the black western blot band for fibronectin (in B and D) is at the same level as band 1 in the stained gels (in A and C).
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of 1-D gel electrophoresis (A and C) and western plot strips (B and D) of proteins in urine samples collected from cats with IdC (A and B) and cats with no urinary tract disease or abnormalities (controls; C and D). The 1-D gel electrophoresis gels for urine samples from a representative cat with IdC and a representative control cat (A and C, respectively) and the western blot strips for urine samples from the same representative cat with IdC and the same representative control cat (B and D, respectively) are illustrated. The 1-D gels were stained with Coomassie blue. Proteins bands were analyzed, and 13 proteins (1 to 13) were identified by means of MALDI-TOF–TOF. The left bar represents the marker (prestained protein laddery). Band number 1 was identified as fibronectin. The positioning of the black western blot band for fibronectin (in B and D) is at the same level as band 1 in the stained gels (in A and C).
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of 1-D gel electrophoresis (A and C) and western plot strips (B and D) of proteins in urine samples collected from cats with IdC (A and B) and cats with no urinary tract disease or abnormalities (controls; C and D). The 1-D gel electrophoresis gels for urine samples from a representative cat with IdC and a representative control cat (A and C, respectively) and the western blot strips for urine samples from the same representative cat with IdC and the same representative control cat (B and D, respectively) are illustrated. The 1-D gels were stained with Coomassie blue. Proteins bands were analyzed, and 13 proteins (1 to 13) were identified by means of MALDI-TOF–TOF. The left bar represents the marker (prestained protein laddery). Band number 1 was identified as fibronectin. The positioning of the black western blot band for fibronectin (in B and D) is at the same level as band 1 in the stained gels (in A and C).
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
MS—To define the identity of the urine proteins, all candidate proteins were analyzed via tandem MS (Table 1). All 13 bands were unambiguously identified and corresponded to 5 proteins. Bands 2 to 7 and 9 and 10 were identified as serum albumin. Band 8 corresponded to haptoglobin, and band 11 corresponded to apolipoproteins A to I. Bands 12 and 13 represented hemoglobin subunits α and β, respectively. Fibronectin was identified as band 1 (Figure 1).
Identification of protein bands separated during 1-D gel electrophoresis of a urine sample collected from 1 of 18 cats with IdC as a result of MALDI-TOF-TOF peptide mass fingerprinting and MS–MS with a tandem mass spectrometer.
| Spot ID | Protein name | Genus and species | Accession No. | MW(Da) | P/ | Protein score |
|---|---|---|---|---|---|---|
| 1 | Fibronectin | Bos taurus | Q95KV5_BOVIN | 38,320 | 7.11 | 85 |
| 2 | Albumin | Felis sylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 117 |
| 3 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 200 |
| 4 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 113 |
| 5 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 461 |
| 6 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 142 |
| 7 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 132 |
| 8 | Haptoglobin | Pongo pygmaeus | HPT_PONPY | 38,471 | 6.79 | 132 |
| 9 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 131 |
| 10 | Albumin | Fsylvestris catus | ALBU_FELCA | 68,615 | 5.46 | 84 |
| 11 | Apolipoprotein A to I | Canis lupus familiaris | APOA1_CANFA | 30,178 | 5.20 | 173 |
| 12 | Hemoglobin subunit β | Erinaceus europaeus | HBB_ERIEU | 15,795 | 8.57 | 93 |
| 13 | Hemoglobin subunit α | Fsylvestris catus | HBA_FELCA | 15,296 | 7.89 | 285 |
The spot ID is the number of the excised band used for identification. The accession number is used for complete information about the protein on a curated protein sequence database.23,k The protein score is identical with the molecular weight search score determined by use of a database search enginei; that score is the negative common logarithm for the probability that the hits are an incidental event. If the score for a certain protein is significant, it is regarded as identified. On the basis of the probability score, theoretical molecular weight and pH are calculated.
ID = Identification. MW = Molecular weight. p/= Isoelectric point.
Western blotting—Macroscopically, fibronectin bands in western blots were much more intense for cats in the IdC group than for cats in the control group (Figure 1), on the basis of quantification of western blot signal intensities with image analysis softwareq (Figure 2). Signal intensities were significantly (P < 0.001) higher for cats with IdC (mean ± SD intensity, 194, 813 ± 184, 610 pixels), compared with the findings for controls (mean intensity, 73, 018 ± 58, 161 pixels). All but 4 cats with IdC had very high amounts of fibronectin in their urine, corresponding to signal intensities > 150, 000 pixels. Of those 4 cats with lower intensities, the signal intensity for urine fibronectin was less than the mean intensity in the controls in only 1 cat. Much lower quantities of urine fibronectin were detected in the urine of controls, corresponding to signal intensities < 100, 000 pixels. Signal intensities in cats with UTI and urolithiasis also were < 100, 000 pixels, except in 1 cat with UTI and 2 cats with urolithiasis. Comparison of signal intensities for controls and cats with UTI (P = 0.79) or urolithiasis (P = 0.81) revealed no significant difference (Figure 3). Comparison of signal intensities for obstructed and nonobstructed cats in the IdC group revealed no significant (P = 0.73) difference (Figure 4). Moreover, comparison of albumin and fibronectin signal intensities was performed in cats with IdC to evaluate the possibility that fibronectin was derived from plasma in hematuric urine, but no significant (P = 0.7) association could be detected.
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with IdC and 18 cats with no urinary tract disease or abnormalities (control group). Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). In the box-and-whisker plots of urine fibronectin signal intensity, the horizontal line in each box represents the median value; the upper and lower boundaries of each box represent the 75th and 25th percentiles, respectively. Whiskers represent the minimum and maximum values. The amount of fibronectin in the urine samples collected from cats with IdC was significantly (P < 0.001) greater than that in urine samples collected from control cats. The photographs over each plot represent representative western blot bands for urine fibronectin in a cat in each group.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with IdC and 18 cats with no urinary tract disease or abnormalities (control group). Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). In the box-and-whisker plots of urine fibronectin signal intensity, the horizontal line in each box represents the median value; the upper and lower boundaries of each box represent the 75th and 25th percentiles, respectively. Whiskers represent the minimum and maximum values. The amount of fibronectin in the urine samples collected from cats with IdC was significantly (P < 0.001) greater than that in urine samples collected from control cats. The photographs over each plot represent representative western blot bands for urine fibronectin in a cat in each group.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with IdC and 18 cats with no urinary tract disease or abnormalities (control group). Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). In the box-and-whisker plots of urine fibronectin signal intensity, the horizontal line in each box represents the median value; the upper and lower boundaries of each box represent the 75th and 25th percentiles, respectively. Whiskers represent the minimum and maximum values. The amount of fibronectin in the urine samples collected from cats with IdC was significantly (P < 0.001) greater than that in urine samples collected from control cats. The photographs over each plot represent representative western blot bands for urine fibronectin in a cat in each group.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with no urinary tract disease or abnormalities (control group), 12 cats with UTI, and 12 cats with urolithiasis. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant differences in median signal intensity for urine fibronectin between the control group and the UTI group (P = 0.79) or the urolithiasis group (P = 0.81) were detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with no urinary tract disease or abnormalities (control group), 12 cats with UTI, and 12 cats with urolithiasis. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant differences in median signal intensity for urine fibronectin between the control group and the UTI group (P = 0.79) or the urolithiasis group (P = 0.81) were detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 18 cats with no urinary tract disease or abnormalities (control group), 12 cats with UTI, and 12 cats with urolithiasis. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant differences in median signal intensity for urine fibronectin between the control group and the UTI group (P = 0.79) or the urolithiasis group (P = 0.81) were detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 10 cats with IdC that also had urethral obstruction and 8 cats with IdC that did not have urethral obstruction. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant (P = 0.73) difference in median signal intensity for urine fibronectin between the cats with IdC and urethral obstruction and the cats with IdC and no urethral obstruction was detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 10 cats with IdC that also had urethral obstruction and 8 cats with IdC that did not have urethral obstruction. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant (P = 0.73) difference in median signal intensity for urine fibronectin between the cats with IdC and urethral obstruction and the cats with IdC and no urethral obstruction was detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Results of western blot analysis to determine the amount of fibronectin in urine samples collected from 10 cats with IdC that also had urethral obstruction and 8 cats with IdC that did not have urethral obstruction. Western blot strips were processed with polyclonal anti–fibronectin antibody; amounts of fibronectin in urine samples were assessed on the basis of fluorescence signal intensity of the western blot bands (evaluated by use of image analysis software). No significant (P = 0.73) difference in median signal intensity for urine fibronectin between the cats with IdC and urethral obstruction and the cats with IdC and no urethral obstruction was detected. See Figure 2 for key.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Histologic examination and immunohistochemical analysis of urinary bladder tissue—Histologic examination of full-thickness urinary bladder biopsy specimens obtained from 6 additional cats (2 with obstructive IdC and 4 controls) revealed loss of bladder wall layering in cats with obstructive IdC (Figure 5). The superficial transitional cell epithelium was injured or even absent in some areas. Subepithelial, intramucosal, and intramuscular bleeding and edema were evident. Fibrosis was present in the bladder wall muscle layer and vasculature. Immunohistochemical analysis of control biopsy specimens revealed fibronectin in the extracellular matrix of the urothelial tissue, especially the subepithelial, intramucosal, and intramuscular tissue. In bladder biopsy specimens from cats with IdC, the distribution pattern of fibronectin was altered; there was only little remaining subepithelial fibronectin, and obvious loss was visible in the submucosal and muscular tunices.
Photomicrographs of sections of full-thickness urinary bladder biopsy specimens obtained from a cat with no urinary tract disease or abnormalities (control group [A and C]) and a cat with obstructive IdC (B and D) and stained with Masson-Goldner stain for histologic examination (A and B) and with fluorescent anti–fibronectin antibody for immunohistochemical detection of fibronectin (C and D). In panels A and B, the green color represents mucus and connective tissue, the light red color represents musculature, the red color represents erythrocytes, the orange to slight red color represents cytoplasm, and the brown to black color represents cell nuclei. In the section from a cat with IdC (B), notice the loss of the physiologic structure of the bladder wall, compared with findings in the section from a control cat (A). In panels C and D, the bright red color represents fibronectin; the blue color is generated by uptake of 4′, 6-diamidino-2-phenylindol, which was also used during immunohistochemical analysis to stain cell nuclei. In the section from a cat with IdC (D), notice the loss of fibronectin from its normal subepithelial, submucosal, and muscular locations illustrated in the section from a control cat (C). a = Transitional cell epithelium. b = Lamina propria. a + b = Mucosal tunic. c = Submucosal tunic. d = Muscle tunic. e = Normal vessel. f = Loss of transitional cell epithelium. g = Intramucosal bleeding. h = Fibrosis in muscle tunic. i = Vascular fibrosis. k = Area of fibronectin loss from subepithelial extracellular matrix.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Photomicrographs of sections of full-thickness urinary bladder biopsy specimens obtained from a cat with no urinary tract disease or abnormalities (control group [A and C]) and a cat with obstructive IdC (B and D) and stained with Masson-Goldner stain for histologic examination (A and B) and with fluorescent anti–fibronectin antibody for immunohistochemical detection of fibronectin (C and D). In panels A and B, the green color represents mucus and connective tissue, the light red color represents musculature, the red color represents erythrocytes, the orange to slight red color represents cytoplasm, and the brown to black color represents cell nuclei. In the section from a cat with IdC (B), notice the loss of the physiologic structure of the bladder wall, compared with findings in the section from a control cat (A). In panels C and D, the bright red color represents fibronectin; the blue color is generated by uptake of 4′, 6-diamidino-2-phenylindol, which was also used during immunohistochemical analysis to stain cell nuclei. In the section from a cat with IdC (D), notice the loss of fibronectin from its normal subepithelial, submucosal, and muscular locations illustrated in the section from a control cat (C). a = Transitional cell epithelium. b = Lamina propria. a + b = Mucosal tunic. c = Submucosal tunic. d = Muscle tunic. e = Normal vessel. f = Loss of transitional cell epithelium. g = Intramucosal bleeding. h = Fibrosis in muscle tunic. i = Vascular fibrosis. k = Area of fibronectin loss from subepithelial extracellular matrix.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Photomicrographs of sections of full-thickness urinary bladder biopsy specimens obtained from a cat with no urinary tract disease or abnormalities (control group [A and C]) and a cat with obstructive IdC (B and D) and stained with Masson-Goldner stain for histologic examination (A and B) and with fluorescent anti–fibronectin antibody for immunohistochemical detection of fibronectin (C and D). In panels A and B, the green color represents mucus and connective tissue, the light red color represents musculature, the red color represents erythrocytes, the orange to slight red color represents cytoplasm, and the brown to black color represents cell nuclei. In the section from a cat with IdC (B), notice the loss of the physiologic structure of the bladder wall, compared with findings in the section from a control cat (A). In panels C and D, the bright red color represents fibronectin; the blue color is generated by uptake of 4′, 6-diamidino-2-phenylindol, which was also used during immunohistochemical analysis to stain cell nuclei. In the section from a cat with IdC (D), notice the loss of fibronectin from its normal subepithelial, submucosal, and muscular locations illustrated in the section from a control cat (C). a = Transitional cell epithelium. b = Lamina propria. a + b = Mucosal tunic. c = Submucosal tunic. d = Muscle tunic. e = Normal vessel. f = Loss of transitional cell epithelium. g = Intramucosal bleeding. h = Fibrosis in muscle tunic. i = Vascular fibrosis. k = Area of fibronectin loss from subepithelial extracellular matrix.
Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1407
Discussion
Diagnostic evaluation and treatment of IdC in cats are often frustrating for owners and veterinarians. Therefore, there is need for research into the cause of IdC and new diagnostic approaches. In human medicine, urine biomarkers for ItC have been investigated, but to the authors' knowledge, no studies to date have been carried out to characterize the proteinuria that develops in cats with IdC. In the present study, 1-D gel electrophoresis revealed that the urine protein pattern in cats with IdC was quite uniformly changed, compared with the pattern in control cats. Although the protein pattern within the control group was also uniform, there were clear differences between patterns in cats with IdC and controls. All urine protein bands in an electrophoresis gel for 1 representative cat with IdC were excised and analyzed by means of MALDI-TOF–TOF. In the urine of that cat with IdC, albumin was detected (Figure 1; bands 2 to 7 and 11 to 12), and increased expression of several albumin fragments was observed. The function of these fragments is unknown, so the importance of their increased expression remains unclear.24 Increased amounts of albumin fragments in the urine of cats with IdC may indicate increased proteolytic activity in cats with this disease. Because albumin is the main plasma protein,25 albumin in urine samples that also contain erythrocytes is likely derived from blood. Thus, assuming that there is an increase in proteolytic activity in the inflamed urinary bladder in cats with IdC, an increase in the fragmented albumin content of urine could likely be the result of proteolysis of albumin within the bladder. An increase in proteolysis could also be one of the reasons for the catabolic state (indicated by the presence of marked tissue lesions) of urinary bladders in cats with IdC. Band 8 was present with similar intensity in the 1-D electrophoresis gels for both cats with IdC and control cats and was identified as haptoglobin. Haptoglobin is a plasma protein,26 and because it was found in both the IdC and control groups, it was probably either filtered from the circulation via the kidneys or produced by the kidneys and excreted into the urine and has no pathological meaning. Apolipoprotein A to I (band 11) is a major protein of plasma high-density lipoproteins synthesized in the liver. In the present study, band 11 was found in the urine of cats with IdC as well as control cats. Apolipoprotein A to I is normally present in urine, and in humans, excretion is only decreased in patients with renal function deficits.27 Bands 12 and 13 corresponded to hemoglobin subunits α and β, respectively. Hemoglobin subunits are part of hemoglobin,28 the iron-containing metalloprotein in RBCs. The presence of those proteins was likely a consequence of hematuria. Hematuria is a common finding in cats with IdC but may be a result of any lower urinary tract disease.29
The protein in the urine samples that was of greatest interest was fibronectin (Figure 1; Table 1); the urine fibronectin content was increased in cats with IdC, compared with that in control cats. This finding suggests a possible role of fibronectin in the pathogenesis of IdC in cats. From studies of humans with urinary bladder cancer, it is known that fibronectin is a 440-kDa glycoprotein30 found in a soluble plasma form and an insoluble matrix form in basement membranes and the extracellular matrix of all tissues in the body.31 It has an important role in cell adhesion, migration, growth, and differentiation32 and is involved in each phase of wound healing31 and clot stabilization.33 Because hematuria was a consistent finding in all cats with IdC, an initial thought was that sanguineous urine could be the source of fibronectin. However, comparison of signal intensities for albumin and fibronectin in cats with IdC revealed no significant association. In addition, cats with UTI or urolithiasis did not have increased fibronectin concentrations in their urine, although most had marked hematuria. Therefore, it is unlikely that blood in the urine was the source of the urine fibronectin.
Fibronectin is present in areas of tissue fibrosis and inflammation,33 and in human medicine, results of several studies30,34,35 suggest urinary fibronectin as a tumor marker. Fibronectin expression is upregulated in patients with urinary bladder neoplasia.36 In those patients, tumor-derived proteases are likely to increase fibronectin release from the urothelial matrix37 during the multistep process of tumor invasion. The concentration of fibronectin can then be measured in the urine by use of an automatic assay. There are also low but measurable amounts of fibronectin in urine of healthy individuals, which is believed to be due to normal suburothelial matrix turnover.36 In human and veterinary medicine, increases in amounts of insoluble fibronectin in urine have been associated with fibrosis38 (eg, renal interstitial fibrosis in rats,39 lung fibrosis in dogs,38 cystic fibrosis in humans,40 and pulmonary fibrosis in rats41) invasion of bladder tumors in humans,34–36 and severe inflammation such as ulcerative colitis in mice.42 Uncontrolled differentiation of fibroblasts into myofibroblasts has been suggested as the primary cause of fibrotic disease.41
To date, no studies regarding fibronectin involvement in development of ItC in humans or IdC in cats have been performed, to the authors' knowledge. In the present study, measurement of fibronectin concentrations in urine samples obtained from cats with IdC, cats with a healthy urinary tract, cats with UTI, and cats with urolithiasis was performed via analysis of western blot signals, and results indicated that there was a significantly greater amount of fibronectin in the urine of cats with IdC. Results of histologic examination of urinary bladder biopsy specimens from cats with obstructive IdC and control cats suggested that the amount of fibronectin in urine was increased in cats with IdC because of marked fibrosis in the bladder muscle layer and vascular walls. The high urine concentration of fibronectin in cats with IdC suggested that fibronectin might become detached from its location within the bladder wall and leaked into the urine. To clarify the origin of the urine fibronectin, immunohistochemical analysis of bladder biopsy specimens from control cats and cats with obstructive IdC was performed by use of anti–fibronectin antibody. The analysis revealed accumulation of fluorescence-marked anti–fibronectin antibody in the subepithelial layer (basement membrane) as well as in the submucosal and muscular layer of healthy bladder tissue specimens. This distribution of fibronectin in control cats is in accordance with findings of another study.43 However, compared with immunohistochemical findings in bladder tissue from control cats, tissue from cats with IdC had an obvious decrease in red fluorescence (ie, anti–fibronectin antibody) in all wall layers. In cats with IdC, detachment of fibronectin from its typical location in the bladder wall is likely a result of increasing destruction of the bladder wall with denudation of umbrella cells,7 loss of tight junctions, and increased permeability of the urothelium.7 Fibronectin leaks into the urine, thereby increasing the amount of urine fibronectin detected in IdC-affected cats. A proposed mechanism is that fibronectin expression is upregulated in cats with IdC because of fibrosis and that it leaks into the urine as a result of destruction and increased permeability of the urinary bladder wall. Upregulation of fibronectin expression in cats with IdC indicates a more important role of fibrosis in the pathogenesis of IdC than previously thought, and measurement of fibronectin concentration in urine might be valuable as a means with which to distinguish between IdC and other diseases of the lower urinary tract in this species.
A limitation of the present study was the fact that most cats with IdC had urethral obstruction; therefore, fibrosis and, in the same manner, the increase in the amount of fibronectin in the urine could be features of obstruction rather than IdC itself. On the other hand, there was no significant difference between urine fibronectin signal intensities in electrophoresis gels for obstructed versus nonobstructed cats, leading to the presumption that the increase in fibronectin occurs in all cats with IdC, regardless of urethral obstruction. In addition, several cats with UTI and urolithiasis also had urethral obstruction; however, the signal intensities for urine fibronectin in electrophoresis gels for those cats were not different from those in gels for control cats. Moreover, results of this study cannot clarify whether fibrosis and subsequent increase in urine fibronectin concentration are the cause or the result of IdC. Further research in this area should be encouraged.
ABBREVIATIONS
| 1-D | 1-dimensional |
| IdC | Idiopathic cystitis |
| ItC | Interstitial cystitis |
| MALDI-TOF-TOF | Matrix-assisted laser desorption and ionization time of flight (tandem mass spectrometry) |
| MS | Mass spectrometry |
| UTI | Urinary tract infection |
Bouin solution, Sigma, Deisenhofen, Germany.
Paraffin-embedding center, Microm International GmbH, Walldorf, Germany.
Handrefractometer, ATAGO Co, Itabashi, Toyko, Japan.
Combur stick, Roche Diagnostics, Roche Germany Holding GmbH, Grenzach-Wyhlen, Germany.
Bradford reagent, Sigma, Deisenhofen, Germany.
10% SDS-gels, Pierce, Bonn, Germany.
Colloidal Coomassie Blue, Pierce, Bonn, Germany.
ABI 4700 Proteomics Analyzer, Applied Biosystems, Minooka, Ill.
MASCOT [database online]. London: Matrix Science Ltd, 1998. Available at: www.matrixscience.com/search_form_select.html. Accessed Jun, 11 2009.
GPS Explorer, version 3.6, Applied Biosystems, Minooka, Ill.
UniProtKB/Swiss-Prot [database online]. Geneva: Swiss Institute of Bioinformatics, 2003. Available at: us.expasy.org/sprot. Accessed Jun 11, 2009.
MSDB metadatabase [database online]. Redmond, Wash: Microsoft Corp, 2008. Available at: msdn.microsoft.com/en-us/library/ms187112.aspx. Accessed June 2009
MOWSE, Imperial Cancer Research Fund, Cancer Research Technology, London, England.
Anti-fibronectin antibody, rabbit, polyclonal, Abnova, Heidelberg, Germany.
Anti-rabbit IgG antibody, polyclonal, goat, AbD Serotec, Düsseldorf, Germany.
Nitrocellulose membranes, GE Healthcare, Freiburg, Germany.
Image Quant TL, version 2005 1.1, GE Healthcare, Freiburg, Germany.
Image Scanner II, GE Healthcare, Freiburg, Germany.
LabScan, version 5.0, GE Healthcare, Freiburg, Germany.
Alexa 568, Invitrogen, Karlsruhe, Germany.
4′, 6-diamidino-2-phenylindol, Invitrogen, Karlsruhe, Germany.
Leica Microsystem, Leica Instruments GmbH, Bensheim, Germany.
Axiovision software, Carl Zeiss AG, Jena, Germany.
PAST (PAleontological Statistics), version 1.90, Natural History Museum, University of Oslo, Oslo, Norway. Available at: folk.uio.no/ohammer/past/index.html. Accessed Jun 11, 2009
Prestained protein ladder, Fermentas, St Leon Rot, Germany.
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