Proteinuria in dogs with gallbladder mucocele formation: A retrospective case control study

2.1 Clinical pathology and endocrine testing

Each dog underwent a complete physical examination by the attending clinician. Blood was collected by venipuncture and urine by ultrasound-guided cystocentesis after a minimum fasting period of 12 hours. For diagnosis of hyperadrenocorticism, blood was drawn 1-hour after IV injection of synthetic cosyntropin for measurement of serum cortisol concentration as previously described.¹² Postcosyntropin serum cortisol concentrations ≥200 ng/mL were considered consistent with a presumptive diagnosis of hyperadrenocorticism.

For diagnosis of hypothyroidism, a serum sample from each dog was stored at −80°C and collectively submitted on dry ice to the Michigan State University Veterinary Diagnostic Laboratory for measurement of serum total thyroxine (T4), free thyroxine by equilibrium dialysis (FT4), and thyrotropin (TSH) concentrations, and for antibodies against thyroxine (T4AA), triiodothyronine (T3AA), and thyroglobulin (TgAA). A laboratory-based diagnosis of hypothyroidism was defined by low serum total T4 and increased serum TSH concentrations or low serum FT4 concentration as previously proposed.^{9, 22, 23}

2.2 Illness severity scoring

To evaluate the impact of overall systemic illness on proteinuria, all dogs were stratified by disease severity into 4 groups based on a previously described scoring system²⁴ as follows: absent (0) for patients that had no clinical signs of systemic illness, mild (1) for patients with signs of clinical disease but suitable for outpatient care, moderate (2) for patients sick enough to require hospitalization and aggressive treatment, and severe (3) for patients with severe illness requiring intensive care and advanced treatment (including all dogs requiring emergency cholecystectomy).

2.3 Assessment of proteinuria

Urinalyses were performed in the North Carolina State University Clinical Pathology Laboratory by an experienced laboratory technician. Urine specific gravity was measured using a digital refractometer (Palm Abbe Digital Refractometer #PA202, MISCO Cleveland, Ohio). Urine chemical analysis was performed using commercially-available dipsticks (Chemstrip 10, Roche Diagnostics, Mannheim Germany) that were read using a calibrated digital analyzer (Chemstrip Criterion II Urine Analyzer, Roche Diagnostics, Mannheim Germany). For sediment examination, 5 mL of urine was centrifuged for 5 minutes at 1600 rpm. After removal of the supernatant, the sediment was suspended, and a drop examined unstained including an average of 10-15 microscopic fields at both 50× and 400× magnification. When requested by the attending clinician, urine protein and urine creatinine concentrations were measured using a clinical chemistry analyzer (Roche Cobas c501, Roche Diagnostics, Mannheim Germany).

Because of the retrospective nature of the study, direct quantitation of urine protein and creatinine concentrations was not performed for the majority of dogs unless originally requested by the attending clinician. Accordingly, proteinuria was determined based on results of urine dipstick analysis. Dogs were categorized as nonproteinuric if the dipstick results indicated no or trace protein and proteinuric if the dipstick results indicated protein concentration ≥30 mg/dL (≥1+). Dogs with substantial blood contamination (gross hematuria) were excluded as possibly having postrenal causes of proteinuria.²⁵

To provide a quantitative estimate of proteinuria, a ratio of urine dipstick protein concentration (mg/dL)-to-urine specific gravity (USG) was calculated for each dog as previously described.²⁶ A cutoff for assuming abnormal protein concentrations in urine was defined as a dipstick protein-to-USG ratio of ≥1.5, which represents 1+ proteinuria (30 mg/mL) in urine with a USG of 1.020 as the upper limit of normal.²⁷

2.4 Statistical analysis

A Fisher exact test was used to identify significant differences in the percentage of control dogs and dogs with gallbladder mucocele formation that had clinical pathology test results out of reference range limits. A Mann-Whitney rank sum test was used to identify significant differences in the median values of clinical pathology test results between control dogs and dogs with gallbladder mucocele formation. Among dogs with gallbladder mucocele formation, associations between abnormal clinical pathology test results and presence or absence of proteinuria (defined by urine dipstick protein concentration to USG ratio ≥1.5 vs <1.5) were determined using a Fisher exact test. Associations between median values of clinical pathology test results and presence or absence of proteinuria (defined by urine dipstick protein concentration to USG ratio ≥1.5 vs <1.5) were determined using a Mann-Whitney rank sum test. Statistical analysis was performed using commercially available software (SigmaPlot 12.0, Systat Software, Inc, San Jose, California). To correct for the impact of multiple testing, Benjamini-Hochberg²⁸ corrected P-values were calculated using a false discovery rate of 0.05. All results reported as statistically significant had a P-value of <.05 and Benjamini-Hochberg corrected P-value <.05. The relationship between grade of illness severity and urine dipstick protein concentration to USG ratio among dogs with gallbladder mucocele formation was evaluated by fitting of a simple logistic regression model with protein ratio (≥1.5 vs <1.5) as the response and grade of illness severity as the predictor. This analysis was performing using R version 4.0.2.

3.1 Case-control study population

A total of 30 case-control pairs of dogs from the previously published study¹² had medical records screened for inclusion in the present study. Five case-control pairs were excluded because of lack of urinalysis in 1 or both dogs. Therefore, 25 case-control pairs of dogs met the criteria for inclusion in the study. Dogs were represented by 15 different breeds including 16 Shetland Sheepdogs, 4 American Cocker Spaniels, 4 Bichon Frise, 4 Chihuahuas, and 2 each of the following breeds: American Staffordshire Terrier, Beagle, Border Collie, Cavalier King Charles Spaniel, Cockapoo, Kerry Blue Terrier, Labrador Retriever, Miniature Poodle, Miniature Schnauzer, Pug, and Shih Tzu. There were 27 castrated males and 23 spayed females. The median age was 10 years (range, 6-16 years) and median body weight 9.4 kg (range, 2.9-35.6 kg). There was no statistically significant difference in sex (P = 1), age (P = .17), or body weight (P = .59) between control dogs and dogs with mucocele formation. Illness severity scores of dogs at the time of participation in the study were: 0 (absent) in 12 (48%) dogs, 1 (mild) in 5 (20%) dogs, 2 (moderate) in 6 (24%) dogs, and 3 (severe) in 2 (8%) dogs with mucocele formation. All control dogs had an illness severity score of 0 (absent). In addition to ultrasonographic diagnosis, gallbladder mucocele formation was confirmed by gross examination and histopathology of gallbladder tissue in 8 (32%) dogs, obtained at the time of surgery (5 dogs) or at necropsy (3 dogs).

3.2 Proteinuria, urinalysis, and urinary tract ultrasound examination

A significantly larger number of dogs with gallbladder mucocele formation had dipstick-measured urine protein concentration ≥30 mg/dL compared to control dogs (P = .003). The median urine dipstick protein concentration to USG ratio (P = .008) and number of dogs having a urine dipstick protein concentration to USG ratio ≥ 1.5 (P = .005) was significantly higher for dogs with gallbladder mucocele formation compared with control dogs (Table 1 and Figure 1). One dog was not included in the analysis because of lack of recorded USG. A quantitative urine protein-to-creatinine (UPC) ratio was measured in 3 dogs with mucocele formation and ranged from 0.82 to 6.7 (concurrent urine dipstick protein concentration to USG ratio range, 1.76-11.11). A UPC was measured in 4 control dogs and ranged from 0.1 to 0.14 (concurrent urine dipstick protein concentration to USG ratio range, 0-0.7).

Prevalence of microscopic hematuria (P = .10) and presence of casts (P = .34) in the urine was not significantly different between dogs with gallbladder mucocele formation compared to control dogs. No dogs in the study were observed to have gross hematuria or pigmenturia. The number of casts of any given type did not exceed 2 per high power field in any dog (Table 1). Nine dogs (36%) with gallbladder mucocele formation had cylindruria with casts of the following types: hyaline (7 dogs), fine granular (4 dogs), waxy (2 dogs), coarse granular (1 dog), epithelial (1 dog), and fatty (1 dog). Among mucocele dogs with cylindruria, 2 dogs had a single cast type, 6 dogs had 2 cast types, and 1 dog had 3 cast types. Five (25%) of the control group dogs had cylindruria with casts of the following types: hyaline (3 dogs), fine granular (3 dogs), and coarse granular (1 dog). Among control dogs with cylindruria, 3 dogs had a single type and 2 dogs had 2 types of casts. Thirteen dogs had urine pH ≥8 (8 dogs with gallbladder mucocele formation and 5 control dogs). No association was found between presence of urine pH ≥8 and urine dipstick protein concentration to USG ratio ≥ 1.5 among dogs in the study (P = .30).

Quantitative urine cultures were performed in 9 gallbladder mucocele dogs with 1 positive for Escherichia coli on a catheterized sample with an inactive sediment and urine dipstick protein concentration to USG ratio of 0.5. One control dog had an active sediment on a voided sample with urine dipstick protein concentration to USG ratio of 0.8. No urine cultures were performed on control dogs.

Results of abdominal ultrasound examination that included the urinary tract were available for 12 dogs with gallbladder mucocele formation and 3 control dogs. Regarding the lower urinary tract, 1 control dog had small suspected uroliths and a urine dipstick protein concentration to USG ratio of 0.4. One dog with a gallbladder mucocele had mild irregularity and thickening of the apical urinary bladder, urine dipstick protein concentration to USG ratio of 11.1, an inactive urine sediment and negative urine culture. All remaining dogs had no ultrasonographic evidence of lower urinary tract disease. Abnormal ultrasound findings related to the kidneys were described in 9/12 (75%) dogs with gallbladder mucocele formation and included decreased corticomedullary distinction (6 dogs), peridiverticular mineralization (6 dogs), hyperechoic kidney cortices (3 dogs), chronic kidney infarcts (2 dogs), and cortical cysts (1 dog). Changes also were described in the kidneys of 2/3 (67%) control dogs, including decreased corticomedullary distinction (2 dogs), peridiverticular mineralization (2 dogs), and cortical rim sign (1 dog).

3.3 Clinicopathologic comorbidities

After correction for the impact of multiple testing on false discovery, results of CBC and serum biochemical profiles identified significantly higher results for the number of polymorphonuclear leukocytes (P = .01) and bands (P = .004), serum activities of alkaline phosphatase (ALP; P = .001), alanine aminotransferase (ALT; P = .008), γ-glutamyl transferase (GGT; P = .002) and lipase (P = .003), and concentration of total bilirubin (P = .005) in dogs with gallbladder mucocele formation compared to control dogs. A significantly higher number of dogs with mucocele formation had results out of reference range limits for serum activities of ALP (P < .001), GGT (P < .001), and lipase (P = .004; Table 2).

Among dogs with gallbladder mucocele formation, no significant differences were found between median results of any CBC or serum biochemistry variables between dogs having a urine dipstick protein concentration to USG ratio ≥1.5 compared to those having a ratio <1.5. As categorized based on whether or not results were outside of reference range limits, no CBC or serum biochemistry variables were significantly associated with urine dipstick protein concentration to USG ratio ≥1.5 vs <1.5 in dogs with gallbladder mucocele formation (Table 3).

3.4 Endocrinopathy

Among dogs with gallbladder mucocele formation, test results consistent with diagnoses of hyperadrenocorticism or hypothyroidism were obtained for 3 and 5 dogs, respectively. No control dogs met criteria for diagnosis of either endocrinopathy. Postcosyntropin serum concentrations of cortisol in dogs with gallbladder mucocele formation (median, 110 ng/mL; interquartile range, 93-140 ng/mL) did not differ significantly from concentrations measured in control dogs (median, 92 ng/mL; interquartile range, 80-116 ng/mL; P = .06; Table 2). Among dogs with gallbladder mucocele formation, no significant association was found between urine dipstick protein concentration to USG ratio ≥1.5 vs <1.5 and postcosyntropin serum cortisol concentration (P = .51), diagnosis of hyperadrenocorticism (P = .48), or diagnosis of hypothyroidism (P = 1; Table 3).

3.5 Illness severity

Illness severity score was significantly associated with urine dipstick protein concentration to USG ratio. Each unit increase in the severity of illness was associated with a 173% increase in odds of a urine dipstick protein concentration to USG ratio ≥1.5 (P = .04; Figure 2).