Urinary gamma-glutamyl transferase and the degree of renal

dysfunction in 75 bitches with pyometra

De Schepper J , De Cock I , Capiau E
Small Animal Department, University of Ghent, Belgium.
Research in Veterinary Science [1989, 46(3):396-400]
Type: Journal Article
Abstract
In 75 bitches with pyometra single urine samples were examined for gammaglutamyl transferase (gamma-GT), protein, glucose, specific gravity, bacteria,
red blood cells and white blood cells. Serum samples were examined for urea,
creatinine, inorganic phosphate and gamma-GT. Biochemical findings were
compared with the degree of illness (clinical signs). Twenty one bitches had
no signs of renal disease. Seventeen showed only glomerular damage
indicated by proteinuria without signs of proximal tubular damage. Thirty
seven bitches had increased urinary gamma-GT levels, indicating proximal
tubular lesions, which were associated with proteinuria in 35 and renal failure
in 16 of them, and worse clinical findings. In all bitches with pyometra serum
levels of gamma-GT were comparable to values in control bitches. Glomerular
dysfunction seemed to precede proximal tubular lesions, so that gamma-GTuria in bitches with pyometra was not an early but rather a late indication of
a more profound degree of renal dysfunction, that is, proximal tubular renal
damage developed after glomerular dysfunction and preceding renal failure.

Clinical presentation of canine pyometra and mucometra:

review
S.D. Pretzercorrespondenceemail
Abilene Animal Hospital, P.A., 320 NE 14th Street, Abilene, KS 67410, US
Abstract;Cystic endometrial hyperplasia (CEH) in the bitch can result in either
pyometra, hematometra, or hydrometra, and many facets of these uterine
diseases can make them difficult to differentiate. The conditions differ in their
systemic effects, since pyometra, particularly closed-cervix pyometra, can be
a life-threatening condition that must be recognized, managed, and treated
expeditiously. Mucometra is an accumulation of sterile intraluminal mucoid
fluid, hematometra is an accumulation of sterile, bloody fluid, and
hydrometra is an accumulation of sterile, watery fluid; none of which have
any significant systemic outward clinical signs. This paper will describe the
definitions, signalment, historical findings, incidence, clinical signs, physical
exam findings, and diagnostic findings in canine pyometra and mucometra,

and hematometra and hydrometra.

;creatinine;dog;renal disease
Creatinine is the analyte most frequently measured in human and veterinary
clinical chemistry laboratories as an indirect measure of glomerular filtration
rate (GFR). Although creatinine metabolism and the difficulties of creatinine
measurement have been reviewed in human medicine, similar reviews are
lacking in veterinary medicine. The aim of this review is to summarize
information and data about creatinine metabolism, measurement, and
diagnostic significance in the dog. Plasma creatinine originates from the
degradation of creatine and creatine phosphate, which are present mainly in
muscle and in food. Creatinine is cleared by glomerular filtration with
negligible renal secretion and extrarenal metabolism, and its clearance is a
good estimate of GFR. Plasma and urine creatinine measurements are based
on the nonspecific Jaff reaction or specific enzymatic reactions; lack of assay
accuracy precludes proper interlaboratory comparison of results.
Preanalytical factors such as age and breed can have an impact on plasma
creatinine (P-creatinine) concentration, while many intraindividual factors of
variation have little effect. Dehydration and drugs mainly affect P-creatinine
concentration in dogs by decreasing GFR. P-creatinine is increased in renal
failure, whatever its cause, and correlates with a decrease in GFR according
to a curvilinear relationship, such that P-creatinine is insensitive for detecting
moderate decreases of GFR or for monitoring progression of GFR in dogs with
severely reduced kidney function. Low sensitivity can be obviated by
determining endogenous or exogenous clearance rates of creatinine. A
technique for determining plasma clearance following IV bolus injection of
exogenous creatinine and subsequent serial measurement of P-creatinine
does not require urine collection and with additional studies may become an
established technique for creatinine clearance in dogs.

Calculation of urinary enzyme excretion, with renal structure

and function in dogs with pyometra
R. HEIENE, L. MOE f1, G.
The urinary enzyme markers of renal damage, alkaline phosphatase (AP), glutamyl transferase (GGT) and N-acetyl--glucosaminidase (NAG),
glomerular filtration rate (GFR) and renal biopsies were studied to evaluate
renal status in dogs with pyometra. After ovariohysterectomy, urinary
enzymes were measured daily for 12 days in 55 dogs, and again at a later
follow-up visit. Thirteen dogs had high levels of at least one enzyme at initial
presentation. Seventeen dogs had a transient increase in urinary enzyme
values between one and five days after surgery. Enzyme values usually
declined to low activities within 12 post-operative days. Renal biopsies
demonstrated tubular abnormalities in many dogs. Mean GFR was 24 and

20ml min1kg1, respectively on day 1 post-operatively and at the followup visit 14 months later. High urinary enzyme values often reflected
extensive lesions in renal proximal tubular cells and sometimes reduced

urine N-acetyl--D-glucosaminidase and -glutamyl

transpeptidase activities in clinically normal adult dogs
Jill D. Brunker, DVM; Nicole M. Ponzio, DVM; Mark E. Payton, PhD
Department of Veterinary Clinical Sciences, Center for Veterinary Health
Sciences, Oklahoma State University, Stillwater, OK 74078. (Brunker, Ponzio);
Department of Statistics, College of Arts and Sciences, Oklahoma State
University, Stillwater, OK 74078. (Payton)
The authors thank Dr. Jerry R. Malayer and the research advisory council,
Center for Veterinary Health Sciences, for funding.
Address correspondence to Dr. Brunker.
ObjectiveTo establish reference ranges for indices of urine N-acetyl-B-Dglucosaminidase (NAG) and G-glutamyl transpeptidase (GGT) activities in
clinically normal adult dogs.
Animals38 dogs.
ProceduresEach dog underwent a physical examination, CBC, serum
biochemical analysis, urinalysis, and serologic testing for heartworm antigen
and antibodies against Ehrlichia canis and Borrelia burgdorferi. Activities of
NAG and GGT in urine were evaluated, and values of the respective indices
were determined as urine NAG or GGT activity (U/L) divided by urine
creatinine concentration (g/L).
ResultsAll dogs were considered clinically normal. A 90% prediction interval
based on the 5th and 95th percentiles for GGT and NAG index values from
both sexes was used to establish the reference ranges for dogs: 1.93 to 28.57
U/g and 0.02 to 3.63 U/g, respectively. Between males and females, urine
NAG index differed significantly, whereas urine GGT index did not. When
accounting for sex differences, reference ranges for the urine NAG index in
males and females were 0.02 to 3.65 U/g and 0.02 to 2.31 U/g, respectively.
Changes in urine pH significantly affected the urine GGT index but not the
urine NAG index. Neither index changed significantly with changes in body
surface area.
Conclusions and Clinical RelevanceData suggest that increases in urine NAG
and GGT indices allow for earlier detection of renal tubular damage in dogs.
Such early detection would enable adjustment of the clinical management of
affected dogs to decrease morbidity and death rates associated with acute

tubular injury and acute tubular necrosis.

Urinalysis to exclude and monitor nephrotoxicity

Robert G Price, Abstract
A large number of compounds, which are in common usage in industry and
medicine, are potentially nephrotoxic. Renal damage and disease resulting
from toxic exposure is progressive and will, if unarrested, culminate in
irreversible renal disease. There is, therefore, a need to develop a battery of
tests with which to monitor and characterise the nephrotoxic cascade. A
European-wide study compared biomarker profiles of adult male workers who
were exposed to heavy metals or solvents. It became apparent that the
urinary profiles varied with the nature of the toxin, reflecting the functional
region of the kidney affected and also the severity of the damage. Children
are a particularly vulnerable group and the investigation of range of
biomarkers indicated that they were indeed susceptible to nephrotoxic
pollutants in their environment. It is proposed that a small cohort of tests
should be used to monitor the early (pre-clinical stages) of renal damage or
dysfunction; these can be supplemented if necessary by additional specific
tests. In the future better information on at-risk populations and genetic
information will enable the determination of individual susceptibility to be
assessed more precisely.
Keywords; Biomarkers; Nephrotoxicity; Sequence of kidney damage; Early
markers; Urine analysis; Battery of tests

Urinary Markers in Healthy Young and Aged Dogs and Dogs

with Chronic Kidney Disease
P.M.Y. Smets1, E. Meyer2, B.E.J. Maddens2, L. Duchateau3 andS. Daminet1
Abstract;Background: Blood urea nitrogen and serum creatinine
concentrations only detect a decrease of >75% of renal functional mass.
Therefore, there is a need for markers that allow early detection and
localization of renal damage.
Hypothesis: Urinary albumin (uALB), C-reactive protein (uCRP), retinol binding
protein (uRBP), and N-acetyl--d-glucosaminidase (uNAG) concentrations are
increased in dogs with chronic kidney disease (CKD) compared with healthy
controls and in healthy older dogs compared with young dogs.
Animals: Ten dogs with CKD, 10 healthy young dogs (age 13 years), and 10
healthy older dogs (age > 7 years) without clinically relevant abnormalities
on physical examination, hematology, biochemistry, and urinalysis.

Methods: Urinary markers were determined using an ELISA (uALB, uCRP, and
uRBP) or a colorimetric test (uNAG). Results were related to urinary creatinine
(c). The fixed effects model or the Wilcoxon rank sum test were used to
compare the different groups of dogs.
Results: uALB/c, uRBP/c, and uNAG/c were significantly higher in CKD dogs
than in healthy dogs. No significant difference was found for uCRP, which was
not detectable in the healthy dogs and only in 3 of the CKD dogs. Between
the healthy young and older dogs, no significant difference was detected for
any of the markers.
Conclusion: The urinary markers uALB/c, uRBP/c, and uNAG/c were
significantly increased in dogs with CKD compared with healthy controls.
Additional studies are needed to evaluate the ability of these markers to
detect renal disease before the onset of azotemia.
Abbreviations:BUN-blood urea nitrogen
CKD-chronic kidney disease
sCr-serum creatinine
uALB/c-urinary albumin-to-creatinine ratio
uCRP/c- urinary C-reactive protein-to-creatinine ratio
uNAG/c-urinary N-acetyl--d-glucosaminidase-to-creatinine ratio
UPC- urinary protein-to-creatinine ratio
uRBP/c-urinary retinol binding protein-to-creatinine ratio
Chronic kidney disease (CKD) is an important cause of morbidity and
mortality in dogs. The prevalence of CKD increases with age, with 15% of
dogs over 10 years old being affected.1 Early diagnosis may allow
therapeutic intervention that prevents further damage and progressive
decline of renal function. However, only a decrease of >75% of renal
functional mass will be detected by current diagnostic tests such as blood
urea nitrogen (BUN) and serum creatinine (sCr) concentrations.2
Unlike these insensitive serum tests, urinary markers are sensitive indicators
of renal injury and also have the potential to reflect the site and severity of
damage.3 They include proteins categorized according to their molecular
weight: high molecular weight (HMW), intermediate molecular weight (IMW),
and low molecular weight (LMW) proteins.4 In general, renal pathologic
proteinuria may be because of increased glomerular filtration or tubular
dysfunction causing impaired reabsorption of normally filtered protein.2
Glomerular dysfunction leads to higher filtration of IMW proteins such as
albumin and in more advanced stages to the presence of HMW proteins in the

ultrafiltrate.4 A urinary albumin (uALB) concentration above normal, but

below the limit of detection (LOD) of conventional dipstick analysis (ie,
between 1 and 30 mg/dL) is defined as microalbuminuria. Several studies
suggest that microalbuminuria may be an early indicator of glomerular
disease in dogs.a,b An example of a HMW protein is C-reactive protein (CRP),
an acute phase protein with an increased serum concentration in many
inflammatory diseases.5 To the authors' knowledge, urinary CRP (uCRP) has
only recently been evaluated in 1 study, reporting an increased CRP-tocreatinine ratio (uCRP/c) in dogs with renal damage secondary to pyometra.c
Tubular dysfunction is reflected by urinary loss of LMW proteins or urinary
enzymes.6 Retinol binding protein (RBP) is a LMW protein associated with
tubular impairment in humans and dogs with renal disease and in
hyperthyroid cats.79 Excessive amounts of urinary enzymes appear in urine
because leakage from damaged tubular cells.10 One such enzyme is Nacetyl--d-glucosaminidase (NAG), a lysosomal glycosidase present in
proximal tubular cells. Urinary NAG (uNAG) has proven to be a useful tool in
early detection of renal injury in various human diseases as reviewed by
Skalova.11 In veterinary medicine, an increased uNAG-to-creatinine ratio
(uNAG/c) has been observed in dogs in various stages of CKD,12,13
leishmaniasis,14 and pyometra13,15 and in dogs treated with nephrotoxic
antibiotics,16 NSAIDS, or glucocorticoids.17
Most studies in dogs have focused on 1 urinary marker, whereas reports on
the combined measurement of glomerular and tubular markers in healthy
and CKD dogs are scarce. Data about urinary marker assay validation likewise
are scarce, especially for NAG.18 Finally, little information exists on the
urinary concentration of these markers in aging dogs.d Therefore, the aims of
this study were 1st to validate a commercial canine albumin ELISA and NAG
colorimetric test. Secondly, we wanted to compare 2 glomerular markers,
uALB and uCRP, and 2 tubular markers, urinary RBP (uRBP) and uNAG
between healthy and CKD dogs. A 3rd objective was to detect a possible agerelated difference in these markers between healthy young and older dogs.
Material and Methods-Dogs
The current study was performed at the Faculty of Veterinary Medicine, Ghent
University, after approval by the Local Ethical Committee. All owners agreed
to participate in the study and signed an informed consent form.
In this cross-sectional study, 20 staff- and student-owned healthy dogs were
recruited and divided into 2 groups according to their age: a group of young
dogs (age 13 years) and a group of healthy older dogs (7 years). Dogs
were judged healthy based on history, physical examination, CBC,
biochemistry profile, and urinalysis [negative bacterial culture and urine
protein-to-creatinine ratio (UPC) < 0.5. Dogs were excluded when medication
possibly influencing renal function had recently been administered.
Descriptive statistics for both groups of healthy dogs are presented in Table 1.

Table 1. Group descriptive statistics (median, range) for the healthy and
chronic kidney disease (CKD) dogs. Variable Young Dogs Healthy Dogs Older
Dogs Total CKD Dogs
Significant difference (P < .0001).
M, male intact; F, female intact; MN, male neutered; FN, female neutered;
sCr, serum creatinine; BUN, blood urea nitrogen; UPC, urine protein-tocreatinine ratio; USG, urine specific gravity.
Number of dogs

10

10

20

10

Age (years) 2.4 (1.12.9)**

8.3 (710.9)**

Sex

2 M, 4 F, 2 MN, 2 FN

2 M, 5 F, 1 MN, 2 FN
2 M, 4 F, 2 MN, 2 FN

Body weight (kg) 23.8 (8.836.0)

17.4 (4.240.1)
sCr (mg/dL) 0.99 (0.691.24)
(1.3715.94)**

5 (1.110.9) 4.7 (0.510.5)

24.6 (8.739)

0.92 (0.421.10)

4 M, 9 F, 3 MN, 4 FN
24.5 (8.739)

0.94 (0.421.24)** 5.51

BUN (mg/dL)33.99 (26.0143.00)

26.13 (18.9843.00)
43.00)**
362.52 (95.98689.01)**
UPC 0.08 (0.060.11)
3.39)**

0.1 (0.060.47)

29.52 (18.98

0.08 (0.060.47)** 1.9 (0.63

USG 1.040 (1.0081.050)

1.030 (1.0161.049)
1.050)**
1.010 (1.0081.017)**

1.035 (1.008

Ten privately owned dogs with CKD of all ages, breeds, and both sexes were
included. Diagnosis was based on clinical signs compatible with CKD (eg,
polyuria, polydipsia, weight loss, inappetence, vomiting), laboratory findings
such as anemia, azotemia, electrolyte disturbances, and urine specific gravity
(USG) < 1.030. Dogs with CKD were classified according to the International
Renal Interest Society (IRIS) into stages I to IV.19 Exclusion criteria were the
presence of concurrent infectious, neoplastic, or endocrine diseases.
Descriptive statistics for the CKD dogs are presented in Table 1.
Clinical signs were polyuria and polydipsia (8/10), lethargy (6/10), weight loss
(5/10), decreased appetite or anorexia (5/10), vomiting (5/10) and diarrhea
(2/10). Fecal examination of 1 dog with diarrhea indicated a Giardia and
Strongyloides stercoralis infection. Two dogs had positive urine cultures
(Escherichia coli). CKD dogs were IRIS stage I (n= 2), III (n= 2), or IV (n= 6)
and all were proteinuric (UPC > 0.5). The 2 dogs in stage I had sCr
concentrations of 1.39 and 1.37 mg/dL and UPC ratios of 2.33 and 3.06,
respectively. Ultrasound findings were hyperechoic renal cortices and a renal
cortical cyst of 3 mm diameter in 1 dog and decreased kidney size (3.8 cm),

hyperechoic parenchyma, and decreased corticomedullary demarcation in the

other dog. Juvenile renal disease was found as underlying cause for CKD in 4
dogs: a Leonberger, a Boxer, a Bull Mastiff, and a Schapendoes (ages 2.3, 0.7,
2.6, and 0.5 years, respectively). The Boxer had a littermate that had been
euthanized because of end-stage renal disease and the Schapendoes was
diagnosed with polycystic kidney disease.
Concurrent heart disease was present in 1 dog with aortic and pulmonary
valve stenosis (Cairn Terrier) and in 2 dogs with mitral valve disease,
International Small Animal Cardiac Health Council (ISACHC) class Ia (Maltese)
and class II (Cavalier King Charles). Nine dogs were newly diagnosed and
referred to our clinic for further diagnostic work-up. In 1 dog, the diagnosis of
CKD had been made 13 months earlier but the owner had stopped feeding
the renal diet and stopped the angiotensin-converting enzyme (ACE) inhibitor
therapy for several months. Six dogs had not yet received any treatment and
4 dogs had already been treated with antiemetics, gastroprokinetics,
antibiotics, or fluid therapy by their referring veterinarians. The dog with
ISACHC class II mitral valve disease was the only dog on an ACE inhibitor
(benazepril) for 12 months.
Sample Collection
Morning urine samples were collected by cystocentesis (10 mL, 22 G needle).
Urinalysis consisting of dipstick analysis,e USG, UPC, sediment analysis, and
bacterial culture, was performed. After centrifugation (3 minutes at 447 g),
the supernatant was divided into aliquots of 0.5 mL and stored at 80C until
analysis of each marker after thawing on ice.
Laboratory Methods
Because spot urine samples were used, urinary concentration of each
biomarker was related to urinary creatinine (c) and expressed as a ratio.16
Urinary creatinine concentration was measured by the modified Jaff reaction
using picric acid.20
Validation of the Albumin ELISA and NAG Colorimetric Assay
As an indication of assay precision, within-day coefficient of variation (CV)
was calculated from 30 duplicate samples assayed on the same day and
between-day CV from 15 samples assayed on separate days, respectively. In
addition, between-day variation was assessed using Lin's sample
concordance correlation coefficient (Lin's CCC, c), which indicates the
degree of deviation from total agreement between 2 repetitive
measurements of the same sample.21
Accuracy of the assays was assessed by evaluation of linearity under
sequential dilution.22 Urinary concentration in diluted samples was plotted
against dilution factor and linear regression analysis was performed.

Assay sensitivity was determined using the mean and standard deviation
(SD) of blank samples to define the lowest concentration of albumin and NAG
that can be reliably distinguished from a blank measurement. The LOD was
calculated as mean blank result + 2.6 SD.23
Albumin, CRP, and RBP ELISA
uALB and uCRP concentration were measured with commercial caninespecific sandwich ELISA kitsf and uRBP with a human ELISA kit,f previously
validated in our laboratory for use with canine urine.24 A microtiter plate
precoated with affinity-purified antibodies for canine albumin, CRP, or human
RBP was used. Wells were filled with 100 L of diluted urine samples or
standards (albumin, 12.5400 ng/mL; CRP, 3.125200 ng/mL; RBP, 7.8250
ng/mL) and incubated. After several wash steps, peroxidase-labeled albumin,
CRP, or RBP antibody conjugate was added. After incubation and wash steps,
each well was filled with 100 L of tetramethylbenzidine substrate and
incubated. Addition of sulfuric acid stopped the colorimetric reaction and
absorbance was measured with an ELISA plate readerg at a wavelength of
450 nm with 650 nm as a reference. A 4 parameter logistic curve-fitting
programh was used to generate the standard curve and calculate albumin,
CRP, or RBP concentrations in the urine samples.
NAG Colorimetric Assay
uNAG activity was calculated with a colorimetric assay.i The NAG enzyme
hydrolyzes the 4-nitrophenyl-NAG substrate and releases p-nitrophenol (PNP).
Addition of the basic stop solution causes ionization of the PNP to the pnitrophenylate ion. The absorbance of the latter ion was measured at 405 nm
with a plate reader.g NAG activity was calculated using a standard formula
and divided by urinary creatinine concentration to determine the uNAG/c
(U/g) = NAG activity (U/L) / urinary creatinine (g/L).13,16
Statistical Analysis
Analyses were performed with a commercial software program.j For normally
distributed data, the linear model with normally distributed random error
term and dog group (healthy and CKD) and age category (young and older)
as categorical fixed effects was fitted. An F-test was used to compare the 2
dog groups. When the normal distribution assumption did not hold, the
Wilcoxon rank sum test stratified for age category was performed to compare
the CKD dogs with the healthy dogs.
In a 2nd analysis, the young healthy dogs were compared with the old
healthy dogs using the same analysis techniques as above, but only for the
healthy dogs and without including age category as an adjusting covariate.
A global significance level of 5% was used to test, leading to a Bonferroniadjusted significance level of 2.5% for each of the 2 comparisons above.

Correlations between different urinary markers and between the markers and
other variables (sCr, BUN, UPC, and IRIS classification) were determined using
Pearson's correlation coefficient for normally distributed data or Kendall's
for the other data.
ResultsValidation of the Albumin and NAG Assays
Precision and sensitivity of the albumin ELISA and NAG colorimetric assay are
presented in Table 2. Both assays had satisfactory variability with within-day
CV<10% and between-day CV<15%. Moreover, a Lin's concordance
correlation coefficient of >0.95 indicated substantial agreement between
repetitive measurements of samples on separate days. Regression analysis
after serial dilution of urine samples showed a linear relationship between
albumin concentration or NAG activity and dilution factor (Fig 1).
Table 2. Assay characteristics for canine albumin ELISA and NAG colorimetric
assay. Assay Characteristic
ALB NAG
ALB, albumin ELISA; NAG, N-acetyl--d-glucosaminidase colorimetric assay;
LOD, limit of detection; CV, coefficient of variation; c, Lin's concordance
correlation coefficient.
Sensitivity: LOD

15.1 ng/mL 0.84 U/L

Precision
Within-day CV (%) 5.2

4.9

Between-day CV (%)

12.0

8.1

0.98 0.98

image
Figure 1. Sequential dilution of a urine sample from a dog with CKD. (A)
Urinary albumin concentration (ng/mL) plotted against dilution factor (
106). The linear regression equation was y= 0.8812x 0.3507 (r2= 0.9995).
(B) Urinary NAG activity (U/L) plotted against dilution factor ( 101). The
linear regression equation was y= 1.0296x+ 0.3107 (r2= 0.995). CKD,
chronic kidney disease; NAG, N-acetyl--glucosaminidase.

Urinary Markers
Results for uALB/c, uRBP/c, and uNAG/c in healthy and CKD dogs are
presented in Figure 2. There was a highly significant difference between
healthy and CKD dogs for uALB/c, uRBP/c, and uNAG/c (P < .0001), but not
for uCRP (P= .07). In the healthy dogs, median uALB/c was 7.3 mg/g (range,
1.5296.5) and in the dogs with CKD uALB/c was 1868.6 (range, 12.7

4594.6). None of the healthy dogs had any detectable uCRP, whereas 3 of the
CKD dogs did, with uCRP/c of 84.3, 179.8, and 236.9 g/g. uRBP/c was 0.1
mg/g (range, 00.9) in the healthy dogs whereas dogs with CKD had a median
ratio of 53.4 (range, 6.81372.2). The healthy dogs had a median uNAG/c of
2.2 U/g (range, 1.25.5) and the CKD dogs had a uNAG/c of 5.7 U/g (range,
1.59.5).

image
Figure 2. Scatter plot of uALB/c (mg/g) (A), uRBP/c (mg/g) (B), and uNAG/c
(U/g) (C) in healthy and CKD dogs. All 3 markers were significantly different (P
< .0001) between groups. uALB/c, urinary albumin-to-creatinine ratio;
uRBP/c: urinary retinol binding protein-to-creatinine ratio; uNAG/c, urinary Nacetyl--glucosaminidase-to-creatinine ratio; CKD, chronic kidney disease.
When comparing the healthy young with the healthy older dogs, no
significant differences were found for uALB/c, uCRP/c, uRBP/c, or uNAG/c (P
> .025). uALB/c in the young dogs was 4.7 mg/g (range, 1.546.3) and in the
older dogs 17.8 (range, 3.3296.5). In the young dogs, median uRBP/c was
0.1 mg/g (range, 00.2) and in the older dogs also 0.1 (range, 00.9). Median
uNAG/c was 2.5 U/g (range, 1.65.5) in the young dogs and in the older dogs
it was 2 (range, 1.25.2).
Correlations between different urinary markers and between the markers and
other variables are shown in Table 3. The highest correlations were found
between uALB/c and UPC (r= 0.96, P < .0001), uALB/c and uRBP/c (r= 0.65, P
< .0001), uRBP/c and UPC (r= 0.74, P < .0001), and uRBP/c and IRIS stage
(r= 0.72, P < .0001). uRBP/c also showed moderate correlations to sCr (r=
0.51, P < .0001) and BUN (r= 0.56, P < .0001), and uALB/c to IRIS stage (r=
0.53, P < .025), but not to sCr and BUN. UCRP/c was weakly to moderately
associated with sCr (r= 0.38, P < .025), IRIS stage (r= 0.52, P < .025), and
BUN (r= 0.41, P < .025), but not with UPC (P > .025). UNAG/c was moderately
correlated to IRIS stage (r= 0.49, P < .025), BUN (r= 0.42, P < .025), and UPC
(r= 0.51, P < .025), but not to sCr.
Table 3. Correlations (r) for the urinary markers, sCr, IRIS stage, BUN, and
UPC. sCr
IRIS Stage BUN UPC uALB/c
uCRP/c
uRBP/c
uNAG/c

Significant correlation (P < .025).

Significant correlation (P < .0001).
sCr, serum creatinine; BUN, blood urea nitrogen; IRIS, International Renal
Interest Society; UPC, urine protein-to-creatinine ratio; uALB/c, urinary
albumin-to-creatinine ratio; uCRP/c, urinary CRP-to-creatinine ratio; uRBP/c,
urinary retinol binding protein-to-creatinine ratio; uNAG/c, urinary N-acetyl-d-glucosaminidase-to-creatinine ratio.

uALB/c
r= 0.27
r= 0.53*
r= 0.65**
r= 0.56*

r= 0.35

r= 0.96**

r= 1

uCRP/c
r= 1

r= 0.52*
r=0.02

r= 0.41*

r= 0.32

r= 0.27

r= 0.72**
r= 0.49*

r= 0.56**

r= 0.74**

r= 0.65**

r= 0.42*

r= 0.51*

r= 0.56*

r= 0.38*
r= 0.43*

uRBP/c
r= 0.51**
r= 0.43*
r= 1

uNAG/c
r= 0.33
r= 0.49*
r=0.02
r= 0.49*
r= 1

r= 0.27

Discussion
Results of the present study indicate satisfactory assay characteristics of the
quantitative canine albumin ELISA and NAG colorimetric assay when applied
for evaluation of urine. Furthermore, uALB, uCRP, uRBP concentrations, and
uNAG activity were found to be significantly higher in dogs with CKD
compared with healthy controls. No age-related difference in urinary markers
was detected between young and older healthy dogs.
The assays used in this study were able to measure canine uALB and uNAG in
a linear manner with within- and between-run imprecision of 5.2 and 12%,
and 4.9 and 8.1%, respectively. As a comparison, for uALB ELISA's, other
studies have reported within- and between-run imprecision ranging between
217 and 4.521.7%, respectively.25,26 For the NAG colorimetric assay,
within- and between-run CVs of 2.8 and 11.9% have been reported.27 In
research settings, where samples frequently are analyzed in the same run,
these levels of imprecision are considered acceptable, but for diagnostic
purposes steps to decrease between-run variability should be taken.
In the current study, both quantitative as well as the qualitative aspects of
proteinuria were evaluated by measuring several proteins as candidate
urinary markers for glomerular and tubular damage. Because albuminuria is
the earliest detectable form of proteinuria and all dogs with CKD had a UPC >
0.5, the strong correlation between uALB/c and UPC was expected.28 This
strong correlation also was found in a study in cats with CKD, in which UPC
also was predictive for survival.25
The use of microalbuminuria as a marker of early CKD is supported by several
studies in dogs predisposed to glomerular disease because of a genetic cause
or a heartworm infection.a,b In these dogs, microalbuminuria reflected the
onset of disease more rapidly than the UPC. However, in the present study,
most of the dogs had advanced renal disease and it was not the objective to
prospectively evaluate uALB/c as an early marker of CKD.
This study is the first to describe uCRP concentrations in dogs with CKD. To

the authors' knowledge, increased uCRP/c ratios only have been described in
dogs with pyometra, and uCRP has never been evaluated in humans.c UCRP
was not detected in the healthy dogs and increased uCRP/c ratios were
present in 3 out of 10 CKD dogs. No increase in uCRP/c was detected in the 2
dogs with positive urine cultures or in the dog with a positive fecal
examination. Although no statistically significant difference could be detected
between CKD and control dogs, the presence of uCRP in dogs with CKD still is
an interesting finding. For CRP to appear in urine, its plasma concentration
must be increased and the glomerular barrier must be sufficiently damaged
to allow HMW protein filtration. Thus, 1 possible hypothesis is that the
increased uCRP/c in these 3 CKD dogs reflects an inflammatory response
leading to increased plasma concentrations and subsequent leakage of CRP
through the damaged glomerular barrier.
In humans, inflammation and oxidative stress start early in the process of
failing kidney function and mild increases in CRP concentration are present
even in patients with moderate renal impairment.29,30 Among other causes,
decreased renal clearance of CRP, proinflammatory cytokines such as
interleukin-6 or both, and uremia are suggested reasons for chronic
inflammation in these human patients.31 This might also have been the
cause in 1 dog with increased uCRP/c, which was in an advanced staged of
CKD (IRIS stage IV) with severe clinical signs (eg, vomiting, hemorrhagic
diarrhea). In humans, serum CRP concentration also is a predictor of
cardiovascular disease, such as congestive heart failure.32 Interestingly, 2 of
the dogs with increased uCRP/c were older dogs that also had mitral valve
disease. One dog with aortic and pulmonary valve stenosis had a normal
uCRP/c. To obtain better insight into the role of inflammation in canine CKD,
plasma and urinary concentrations of CRP, and other proinflammatory
mediators should be evaluated in a larger number of dogs.
uRBP was measured as a 1st marker of tubular dysfunction. In most
mammals, this LMW protein circulates in plasma complexed with a 2nd
protein (transthyretin) and binds vitamin A, which prevents RBP excretion.
However, dogs have high concentrations of transthyretin-uncomplexed RBP
that is filtered by the glomeruli. Under physiologic conditions, the filtered RBP
is almost completely reabsorbed by megalin-mediated endocytosis in the
proximal tubular cells, but tubular dysfunction leads to excessive amounts of
uRBP.8,33 In our study, uRBP/c was significantly higher in dogs with CKD
compared with healthy controls and highly correlated with sCr, BUN, UPC, and
IRIS-stage, which corroborates results from previous studies documenting
increased uRBP/c ratios in CKD dogs.8,34
As a 2nd marker for tubular dysfunction, uNAG was determined. Indeed, in
humans increased uNAG/c in renal disease does not originate from filtration
because glomerular damage but from tubular epithelial cells.35,36 This
enzyme was found to be significantly higher in the CKD than in the healthy
dogs, although there was large overlap in uNAG/c ratios between the 2

groups. In the present study, uNAG/c in CKD dogs (median, 5.5 U/g; range,
1.59.5 U/g) was lower than observed in 2 previous reports in 9 CKD dogs
(mean SD, 17.1 7.9 U/g) and 7 CKD dogs (median, 25.4 U/g; range, 15.7
136.8 U/g), respectively.12,13 Possible explanations for this difference include
variation in laboratory techniques for NAG analysis and dog-related factors,
such as stage of CKD. Enzymuria may be a marker of active disease
damaging the renal cells. When the primary cause of the damage has
disappeared, enzymuria might become minimal despite pronounced
structural damage and permanent loss of tubular cells.35 To further
investigate this hypothesis, additional studies are needed to determine
uNAG/c in dogs with acute renal failure as well as in dogs with various stages
of CKD.
Urinary tract infection (UTI) was an exclusion criterion for the healthy dogs,
but 2 of the CKD dogs had positive urine cultures. The effect of lower UTI on
microalbuminuria and uNAG/c seems to be minimal in dogs, but an increased
uNAG/c ratio is reported in the presence of pyelonephritis.13,37 In these 2
dogs, no ultrasonographic signs of pyelonephritis were detected. To the
authors' knowledge, the effect of UTI on uRBP and uCRP is unknown in dogs.
In humans, microalbuminuria, and uCRP, uRBP, and uNAG concentrations are
increased in patients with upper UTI, but not in patients with cystitis or
asymptomatic bacteriuria.3841
The degree of correlation between the urinary markers and routine variables
such as sCr, BUN, and UPC differed for each urinary marker. This is not
unexpected, because these markers might be more sensitive indicators of
renal damage and some may reflect renal dysfunction at another site rather
than serving as an indirect glomerular marker as does sCr. Different markers
each may reflect different pathophysiologic processes. UALB/c was not
correlated with sCr and BUN whereas uRBP/c was strongly correlated with
both. Progression of CKD in humans and dogs, respectively, may be initiated
by glomerular changes subsequently leading to interstitial injury.4,42,43 It is
mainly the tubulointerstitial inflammatory process that precedes renal
scarring and is associated with a decreased renal function. In this more
advanced stage, large amounts of LMW proteins such as RBP appear in urine.
Negatively charged IMW proteins such as albumin are a sign of mildly altered
glomerular permeability and already appear in urine in an early stage.
Although uCRP/c was moderately to weakly correlated to sCr, BUN, and UPC,
this finding needs to be interpreted with caution because only 3 dogs had
positive results. As in the present study, a report on urinary enzymes in
humans with glomerulonephritis also demonstrated a correlation between
uNAG/c and proteinuria and the absence of an association between uNAG/c
and sCr.44 The current hypothesis is that urinary enzymes correlate better
with tubulointerstitial damage than with an indirect measurement of
glomerular filtration rate such as sCr.
In the healthy dogs, a median uALB/c of 7.3 mg/g (range, 1.5296.5) was

measured. Cut-off values for uALB/c ratios remain to be properly established

in a large number of healthy dogs. Usually, a normal range is calculated as
mean uALB/c + 2 SD of the healthy control group.45 In 2 previous reports,
uALB/c either ranged from 5 to 82 mg/g (n= 6) or mean uALB/c SD was 40
80 mg/g (n= 10).45,k Three of 20 healthy dogs in the current study had
uALB/c ratios above the upper limit of 42 mg/g (mean + 2 SD of the healthy
young dogs). Interestingly, 2 of these 3 dogs were older dogs with uALB/c
ratios well above the cut-off (152.6 and 296.5 mg/g) and another 2 older
dogs had uALB/c ratios near the cut-off (40.4 and 41.5 mg/g). Previous data
indicated a higher prevalence of microalbuminuria with increasing age.d This
finding might be related to the higher prevalence of glomerular lesions and
CKD in aging dogs.1,46 However, in the present study no renal biopsies were
taken because ethical considerations. As for the CKD dogs, knowledge of the
histopathologic lesions would unlikely have therapeutic or prognostic
implications in advanced disease stages.
uNAG/c ratios in the healthy dogs (median, 2.2 U/g; range, 1.25.5 U/g) were
in agreement with previous reports.13,27,47 The present study is the first to
report uNAG/c ratios in older healthy dogs. No significant difference was
found between the young and older healthy dogs. Nevertheless, the true
effect of age is best assessed in a longitudinal follow-up of the same
individuals. In humans, uNAG is higher in children than in adults.48 This is
mainly because changes in muscle mass and consequently in creatinine
excretion.49 Therefore, in human medicine each laboratory establishes its
own reference ranges for different age categories and patients then are
compared against age-matched controls.48,50
In conclusion, combined use of glomerular and tubular markers in conjunction
with traditional tests may provide more detailed information about the extent
and location of renal damage. The urinary markers discussed in this study
appear to be promising as noninvasive tools for the diagnosis of CKD in dogs.
Age-related differences seem to be minimal between young adult and older
healthy dogs. These results should encourage in-depth investigation of
urinary markers in larger numbers of dogs classified according to their
etiology and stage of renal disease.
Footnotes
a Grauer GF, Oberhauser EB, Basaraba RJ, et al. Development of
microalbuminuria in dogs with heartworm disease. J Vet Intern Med
2002;16:352 [abstract]
b Lees GE, Jensen WA, Simpson DF, Kashtan CE. Persistent albuminuria
precedes the onset of overt proteinuria in male dogs with X-linked heriditary
nephropaty. J Vet Intern Med 2002;16:353 [abstract]
c Maddens BEJ, Daminet S, Smets P, et al. Urinary immunoglobulin G, Creactive protein and retinol-binding protein as candidate early biomarkers for

renal dysfunction in dogs with pyometra. J Vet Intern Med 2008;22:1473

[abstract]
d Radecki S, Donnelly R, Jensen WA, Stinchcomb DT. Effect of age and breed
on the prevalence of microalbuminuria in dogs. J Vet Intern Med 2003;17:406
[abstract]
e Combur stick, Roche Diagnostics, Mannheim, Germany
f Immunology Consultants Laboratory, Newberg, OR
g Multiskan MS, Labsystems Thermo Fisher Scientific, Waltham, MA
h Deltasoft JV, BioMetallics Incorporated, Princeton, NJ
i Sigma-Aldrich, St Louis, MO
j SAS version 9.1, SAS Institute. Inc, Cary, NC
k Mazzi A, Fracassi F, Gentilini F. Urinary protein to creatinine ratio and
albumin to creatinine ratio in dogs with diabetes mellitus and pituitary
dependent hyperadrenocorticism. Congress Proceedings of the 16th ECVIMCA Congress, Amsterdam, the Netherlands, September 1416, 2006
[abstract]
Acknowledgments
This study was funded by a grant from the Fonds voor Wetenschappelijk
Onderzoek (FWO) Vlaanderen. The authors thank Kristel Demeyere for her
technical assistance.
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Anaemia and leucocytosis in one hundred and twelve dogs

with pyometra
J. DE SCHEPPER, J. VAN DER STOCK andE. CAPIAU
ABSTRACT

In a group of 112 dogs with pyometra, 31 per cent showed a normal red blood
cell picture, 57 per cent non-regenerative normocytic normochromic anaemia
and 12 per cent non-regenerative microcytic hypochromic anaemia. The nonregenerative anaemia was absent or moderate at lower white blood cell
levels and markedly present at high to extreme white blood cell counts. The
degree of non-regenerative anaemia was positively correlated with the
degree of leucocytosis, neutrophilia, left shift and monocytosis. The more
pronounced non-regenerative microcytic hypochromic anaemia, indicating a
more severe chronic blood loss, was usually encountered at extremely high
white blood cell levels. The significance of these observations is discussed.

Calculation of urinary enzyme excretion, with renal structure

and function in dogs with pyometra
R. HEIENE, L. MOEf1,
Abstract
The urinary enzyme markers of renal damage, alkaline phosphatase (Full-size
image (<1 K)), -glutamyl transferase (Full-size image (<1 K)) and N-acetyl-glucosaminidase (Full-size image (<1 K)), glomerular filtration rate (Full-size
image (<1 K)) and renal biopsies were studied to evaluate renal status in
dogs with pyometra. After ovariohysterectomy, urinary enzymes were
measured daily for 12 days in 55 dogs, and again at a later follow-up visit.
Thirteen dogs had high levels of at least one enzyme at initial presentation.
Seventeen dogs had a transient increase in urinary enzyme values between
one and five days after surgery. Enzyme values usually declined to low
activities within 12 post-operative days. Renal biopsies demonstrated tubular
abnormalities in many dogs. Mean Full-size image (<1 K) was 24 and 20ml
min1kg1, respectively on day 1 post-operatively and at the follow-up visit
14 months later. High urinary enzyme values often reflected extensive
lesions in renal proximal tubular cells and sometimes reduced Full-size image
(<1 K).

Clinico-haematological and serum biochemical

alterations in pyometra affected bitches

Amit R Patil, Madhu Swamy, Astha Chandra, Shobha

Jawre
Abstract
Canine pyometra is the infective disease of progesterone influenced uterus of
intact bitches and known to cause a systemic inflammatory responses
leading to multiple systemic dysfunctions. In the study twenty bitches were
divided into two groups after a presumptive diagnosis of pyometra. The
diagnosis was based on history, clinical examination and ultrasonography. Ten
animals were placed into two groups each. Group I comprised of non-surgical
cases receiving medicinal treatment and group II of the surgical cases going
for ovariohysterectomy and blood samples of same animals were obtained
again fifteen days after undergoing treatment either non surgical that is,
medicinal (group III) or surgical (group IV), respectively. Blood was also
collected from ten clinically healthy bitches. Haematological examination
revealed a decrease in the total erythrocyte count (TEC), haemoglobin, and
packed cell volume (PCV) in all pyometra infected animals in comparison to
control value. In this study, the mean haemoglobin content of control animals
was determined to be 13.420.76 g/dl. In group I animals the mean
haemoglobin was determined as 9.322.51 g/dl, which increased after 15
days of treatment to 12.142.14 g/dl. In pyometra infected bitches, the mean
value of haemoglobin was increased 15 days post surgery to 11.071.70 g/dl
from a mean value of 7.382.16 g/dl before surgery and moreover the mean
value of TEC was increased 15 days post surgery to 5.211.14106/l from a
mean value of 3.771.12106/l before surgery. In pyometric animals
undergoing surgical treatment, the mean value of PCV before surgery was
24.186.85%, which 15 days post surgery was increased up to 36.24.87%.
Band cells and Total Leucocyte count (TLC) parameters differed significantly
with their control values. Leucocytosis with neutrophilia was a consistent
finding along with increased number of band cells per microscopic field in
pyometric cases which were decreased after both surgical and medicinal
treatments. Biochemically, there were significant elevations of serum Alanine
transferase (ALT), Aspartate transferase (AST), Alkaline phosphatase (ALP),
Total bilirubin (BIT), Gamma glutamyl transferase (GGT), Blood urea nitrogen
(BUN) and Creatinine (CRE), and decrease in protein and albumin
concentration in serum of all dogs with pyometra. In pyometric animals
undergoing medicinal treatment by drugs the mean value of ALT was
determined as 71.8621.95 IU/L, which decreased to 58.0414.44 IU/L after
treatment. In pyometric animals undergoing ovariohysterectomy, the mean
value of ALT was decreased 15 days post surgery to 39.817.91 IU/L from the
mean value of 99.1644.01 IU/L before surgery. The mean value of AST was
decreased 15 days post ovariohysterectomy to 21.062.19 IU/L from a mean
value of 54.0223.94 IU/L before surgery. In pyometra infected animals
undergoing treatment by drugs the value of ALP was determined as
102.3346.03 IU/L, which decreased to 51.8034.16 IU/L after treatment. An
increase in serum CRE and BUN values were recorded in all cases of

pyometra which reduced to lower levels during both treatments in follow-ups.

All the haemato-biochemical parameters were comparable to their respective
reference values after either medicinal treatment or ovariohysterectomy of
dogs. Thus the dogs with pyometra show significant abnormalities in
haemato-biochemical parameters indicative of reversible nature of liver and
kidney dysfunction in pyometra.

Hysterectomy leads to fast improvement of haematological

and immunological parameters in bitches with pyometra
A. Bartoskova1, R. Vitasek1, L. Leva2 andM. Faldyna1,2
Objectives: To investigate whether a combination of hysterectomy and
antibiotic treatment leads to an improvement of altered haematological and
immunological parameters in bitches affected by pyometra.
Methods: Blood samples obtained from 13 bitches affected by pyometra were
investigated before hysterectomy and seven days later for a total and
differential leucocyte count, activity of neutrophils and lymphocytes, and
quantification of total serum immunoglobulins, lysozyme and circulating
immune complexes.
Results: The parameters most affected included changes in blood profile
(leucocytosis because of neutrophilia or leucopenia) and inhibition of
lymphocyte activity. Seven days after hysterectomy, all affected parameters
returned to normal levels comparable to clinically healthy dogs.
Clinical Significance: Application of immunostimulating therapy is not
necessary to manage changes in blood cell counts and suppression of
lymphocyte activity in bitches with pyometra. Removal of the infected uterus,
being the source of infection, leads to improvement.

open Pyometra
Richard W. Nelson, D.V.M. , , Edward C. Feldman, D.V.M.
Pyometra is a relatively uncommon diestral uterine disorder seen primarily in
older bitches and queens. The incidence of pyometra appears to be
increasing, especially in younger animals, as a result of increasing use of
estrogen and progesterone for mismating and for certain medical disorders.
The clinical signs of pyometra and abnormalities on physical examination are
dependent on the patency of the cervix and how quickly the client recognizes
the problem. Ovariohysterectomy is still the treatment of choice; however,
prostaglandin F2 as a medical alternative in the management of pyometra is
gaining more and more acceptance. The goal of prostaglandin F2 therapy is
to salvage the reproductive tract in the young breeding bitch or queen in the
hopes of obtaining future litters. The results have been very promising,
especially for bitches or queens with open-cervix pyometra. Prostaglandin
F2 appears to offer the owner a reasonable alternative in the management
of pyometra.

Pyometra in the bitch

J. P. RENTON1, T. A. DOUGLAS2 andC. WATTS3

ABSTRACT
The diagnosis of pyometra in the bitch was aided by radiographic
examination of the abdomen and by a total white cell count. In cases of
pyometra described, some uterine distension was seen on radiographic
examination. A study was made of the course of blood urea levels as a guide
to prognosis. High blood urea levels before operation may subsequently
return to normal. The cases with poor prognosis were those in which the
blood urea level rose steadily after ovarohysterectomy. Escherichia coli was
found in almost all cases of pyometra, both closed and open.
There was no evidence of a poorer prognosis in closed than in open cases of
pyometra.

Pyometra
Richard W. Nelson, D.V.M. , , Edward C. Feldman, D.V.M.
pyometra is a relatively uncommon diestral uterine disorder seen primarily in
older bitches and queens. The incidence of pyometra appears to be
increasing, especially in younger animals, as a result of increasing use of
estrogen and progesterone for mismating and for certain medical disorders.
The clinical signs of pyometra and abnormalities on physical examination are
dependent on the patency of the cervix and how quickly the client recognizes
the problem. Ovariohysterectomy is still the treatment of choice; however,
prostaglandin F2 as a medical alternative in the management of pyometra is
gaining more and more acceptance. The goal of prostaglandin F2 therapy is
to salvage the reproductive tract in the young breeding bitch or queen in the
hopes of obtaining future litters. The results have been very promising,
especially for bitches or queens with open-cervix pyometra. Prostaglandin
F2 appears to offer the owner a reasonable alternative in the management
of pyometra.

Renal dysfunction in dogs with pyometra.

Stone EA , Littman MP , Robertson JL , Bove KC
Abstract
Highlight Terms Highlight biological terms.
No biological terms identified
Renal function and pathologic changes in 27 dogs with pyometra were
studied. Evaluation included CBC; serum biochemical evaluation; urinalysis;
urine and uterine bacteriologic culture; uterine morphologic features; and
light, electron, and immunofluorescent microscopic evaluation of renal

tissues. Measurements of 24-hour creatinine clearance, protein excretion, Na

excretion, and urine volume were made in 12 dogs without azotemia. Of 27
dogs, 26% were azotemic and 89% had a urine sp gr less than 1.035.
Glomerular filtration rate was reduced in 75% of 12 dogs without azotemia.
None of these 12 dogs was proteinuric. Examination of renal biopsy
specimens revealed a high prevalence of mild tubulointerstitial nephritis, but
few specific glomerular lesions. Minimal immunofluorescence was detected
within the mesangium in 18% of the dogs. Immunofluorescence was not
associated with the interstitium or tubules. Urinary tract infection was
detected in 22% of the dogs. Escherichia coli and Klebsiella were recovered
from the uterus in 59 and 15% of the dogs, respectively. Low urine specific
gravity values were obtained from dogs without azotemia and from dogs with
uterine cultures considered negative for E coli and other gram-negative
bacteria. The reduction in glomerular filtration rate was a functional
abnormality not correlated with structural damage in the glomerulus.
Reproductive Ultrasound of the Bitch
Autumn P. Davidson, DVM, MS, Dipl ACVIMa, , , Tomas W. Baker, MSb
ultrasonographic evaluation of the reproductive tract is an important
component in the evaluation of the bitch and queen. Information is obtained
concerning normal events involving the reproductive system (eg, ovulation,
pregnancy) as well as pathologic conditions (eg, ovarian cysts, metritis). The
appearance of the female reproductive tract normally changes with phases of
the cycle; these changes need to be interpreted with knowledge of the
ovarian cycle. Serial ultrasonographic evaluation of the diseased reproductive
tract can be very helpful in evaluating response to therapy.

Systemic inflammarory response in canine pyometra the

response to uterine bacterial infection
Fransson, Boel (2003).
Abstract
Research efforts have focused mainly on the hormonal aspects of canine
pyometra for more than 6 decades. However, this disease is often manifested
as systemic illness in response to the bacterial uterine infection. Studies I-II
were undertaken to clarify bacteriological aspects of canine pyometra; i.e. the
origin of the infecting bacteria, the infecting bacterias impact on severity of
the systemic illness and the presence of bacterial endotoxin in the systemic
circulation. Study I, a bacterio-epidemiologic study, investigated the
predominant bacteria, Escherichia coli, using biochemical fingerprinting. The
homogeneity among E. coli populations, isolated from various sites in bitches
with pyometra and from faeces of healthy dogs, was determined. Study II, a
clinical study of bitches with pyometra, determined uterine bacterial species,
haematology, blood biochemical parameters and plasma endotoxin levels.

The impact of infecting bacteria on blood parameters and clinical status was
studied. Study III investigated if bitches with pyometra display the Systemic
Inflammatory Response syndrome (SIRS) and if SIRS relates to outcome.
Systemic levels of interleukin-6 (IL-6), C - reactive protein (CRP) and tumor
necrosis factor (TNF) were determined, investigating a possible correlation
between these inflammatory markers and SIRS or outcome. Study IV used
clinical parameters, haematology, blood biochemical parameters, CRP and
TNF to clinically differentiate pyometra from the often preceding uterine
condition cystic endometrial hyperplasia (CEH). Study I revealed that E. coli
isolated from bitches with pyometra show high level of homogeneity
indicating that E. coli associated with pyometra may have properties, yet
undetermined, in common. Identical clones of E. coli were found in the faeces
and uterus of bitches with pyometra, indicating an ascending infection route.
Study II failed to show systemic endotoxemia in bitches with pyometra, but
showed many other signs of systemic affection in blood parameters. Study III
showed that 57 % of pyometra cases fulfil clinical criteria for SIRS and that
SIRS criteria are correlated to increased length of hospitalization. Body
temperature, heart rate and CRP correlated to SIRS and CRP correlated to
outcome. Study IV revealed that clinical signs and levels of percent band
neutrophils and CRP aid in the differentiation of CEH from pyometra.
ALL I. Wadas (Fransson) B., Khn I., Lagerstedt A-S. & Jonsson P. 1996.
Biochemical phenotypes of Escherichia coli in dogs: Comparison of isolates
isolated from bitches suffering from pyometra and urinary tract infection with
isolates from faeces of healthy dogs. Veterinary microbiology 52: 293-300 II.
Fransson B., Lagerstedt A-S., Hellmen E. & Jonsson P. 1997. Bacteriological
findings, blood chemistry profile and plasma endotoxin levels in bitches with
pyometra or other uterine diseases. Journal of veterinary medicine series A
44: 417-426 III. Fransson B.A., Lagerstedt A.-S., Bergstrom A., Hagman R.,
Park J.S., Chew B.P., Evans M.A. & Ragle C.A. 2003. Systemic inflammatory
response in canine pyometra. Submitted for publication. IV. Fransson B.A.,
Karlstam E., Bergstrom A., Lagerstedt A.-S., Park J.S., Evans M.A. & Ragle C.A.
2003. C-reactive protein can aid in the differentiation of pyometra from cystic
endometrial hyperplasia in dogs. Accepted for publication in Journal of the
American Animal Hospital Association.

Systemic inflammarory response in canine pyometra the

response to uterine bacterial infection
Fransson, Boel (2003).
Abstract
Research efforts have focused mainly on the hormonal aspects of canine
pyometra for more than 6 decades. However, this disease is often manifested
as systemic illness in response to the bacterial uterine infection. Studies I-II
were undertaken to clarify bacteriological aspects of canine pyometra; i.e. the
origin of the infecting bacteria, the infecting bacterias impact on severity of

the systemic illness and the presence of bacterial endotoxin in the systemic
circulation. Study I, a bacterio-epidemiologic study, investigated the
predominant bacteria, Escherichia coli, using biochemical fingerprinting. The
homogeneity among E. coli populations, isolated from various sites in bitches
with pyometra and from faeces of healthy dogs, was determined. Study II, a
clinical study of bitches with pyometra, determined uterine bacterial species,
haematology, blood biochemical parameters and plasma endotoxin levels.
The impact of infecting bacteria on blood parameters and clinical status was
studied. Study III investigated if bitches with pyometra display the Systemic
Inflammatory Response syndrome (SIRS) and if SIRS relates to outcome.
Systemic levels of interleukin-6 (IL-6), C - reactive protein (CRP) and tumor
necrosis factor (TNF) were determined, investigating a possible correlation
between these inflammatory markers and SIRS or outcome. Study IV used
clinical parameters, haematology, blood biochemical parameters, CRP and
TNF to clinically differentiate pyometra from the often preceding uterine
condition cystic endometrial hyperplasia (CEH). Study I revealed that E. coli
isolated from bitches with pyometra show high level of homogeneity
indicating that E. coli associated with pyometra may have properties, yet
undetermined, in common. Identical clones of E. coli were found in the faeces
and uterus of bitches with pyometra, indicating an ascending infection route.
Study II failed to show systemic endotoxemia in bitches with pyometra, but
showed many other signs of systemic affection in blood parameters. Study III
showed that 57 % of pyometra cases fulfil clinical criteria for SIRS and that
SIRS criteria are correlated to increased length of hospitalization. Body
temperature, heart rate and CRP correlated to SIRS and CRP correlated to
outcome. Study IV revealed that clinical signs and levels of percent band
neutrophils and CRP aid in the differentiation of CEH from pyometra.
ALL I. Wadas (Fransson) B., Khn I., Lagerstedt A-S. & Jonsson P. 1996.
Biochemical phenotypes of Escherichia coli in dogs: Comparison of isolates
isolated from bitches suffering from pyometra and urinary tract infection with
isolates from faeces of healthy dogs. Veterinary microbiology 52: 293-300 II.
Fransson B., Lagerstedt A-S., Hellmen E. & Jonsson P. 1997. Bacteriological
findings, blood chemistry profile and plasma endotoxin levels in bitches with
pyometra or other uterine diseases. Journal of veterinary medicine series A
44: 417-426 III. Fransson B.A., Lagerstedt A.-S., Bergstrom A., Hagman R.,
Park J.S., Chew B.P., Evans M.A. & Ragle C.A. 2003. Systemic inflammatory
response in canine pyometra. Submitted for publication. IV. Fransson B.A.,
Karlstam E., Bergstrom A., Lagerstedt A.-S., Park J.S., Evans M.A. & Ragle C.A.
2003. C-reactive protein can aid in the differentiation of pyometra from cystic
endometrial hyperplasia in dogs. Accepted for publication in Journal of the
American Animal Hospital Association.

The Significance of Radiology in the Diagnosis of Pyometra

H. van Bree*, J. de Schepper* andE. Capiau*
131 bitches with proven pyometra or endometritis post oestrum (E. P. O.) a

retrospective study of the lateral abdominal radiograph was carried out. In 31

(24%) of the cases (group 1) the uterine silhouette could not be perceived on
the survey radiograph. In the other 100 cases (group 2) the uterus was
clearly visible. Its diameter was measured and compared to the length of the
second lumbar vertebra. The values for this ratio ranged between 0.4 and
3.5. The smallest uterine diameter perceived on the plain lateral abdominal
radiograph measured 12 mm. Within group 2 the degree of uterine distension
was significantly correlated with the number of white blood cells and the
serum haptoglobin concentration. This correlation suggests a relation
between the degree of uterine distension, the amount of uterine pus, the
degree of uterine inflammation and the general inflammatory and stress
reaction. Renal failure was more frequently observed in bitches with a greatly
enlarged uterus than with a normal or only slightly enlarged uterus, although
the degree of renal failure was not correlated to the uterine diameter. Within
group 2 the closed type of pyometra was associated with a more enlarged
uterus, but not with a higher leucocytosis.

urine protein-to-creatinine ratio in urine samples

Comparison of urine protein-to-creatinine ratio in urine samples collected by
cystocentesis versus free catch in dogs
Laura Beatrice, DMV; Francesca Nizi, DMV; Daniela Callegari, DMV, PhD;
Saverio Paltrinieri, DMV, PhD; Eric Zini, DMV, PhD; Paola D'Ippolito, DMV;
Andrea Zatelli, DMV
ObjectiveTo assess whether urine protein-to-creatinine (UPC) ratios
determined in urine samples collected by cystocentesis versus those
collected by free catch provide similar diagnostic information for dogs.
Animals115 client-owned dogs evaluated because of various health
problems requiring urinalysis or to screen for proteinuria in an area endemic
for leishmaniasis.Procedures230 paired urine samples, 1 collected by
cystocentesis and 1 by free catch, were collected from the 115 dogs. The UPC
ratio was determined in paired urine samples (n = 162) from 81 dogs with no
indication of active inflammation according to urine sediment analysis. On
the basis of the UPC ratio of urine sample collected by cystocentesis, dogs
were classified as nonproteinuric (UPC ratio < 0.2), borderline proteinuric
(UPC ratio of 0.2 to 0.5), or proteinuric (UPC ratio > 0.5), according to the
International Renal Interest Society (IRIS).
ResultsThe correlation between UPC ratio in urine samples collected by
cystocentesis and by free catch was strong (r2 = 0.90); 75 of 81 (92.6%)
dogs had UPC ratios from both urine samples that resulted in classification in
the same IRIS substage with a kappa coefficient of 0.83.
Conclusions and Clinical RelevanceThe UPC ratio in dogs was minimally
affected in urine samples collected by free catch, thus allowing correct

grading of proteinuria with this method. The high reliability of the UPC ratio in
free-catch urine samples coupled with the ease of collection should increase
the use of this value for assessment of proteinuria.
Uropathogenic virulence factors in isolates of Escherichia coli
from clinical cases of canine pyometra and feces of healthy
bitches
Yvette M.M Chena, Patrick J Wrighta, Chee-Seong Leeb, Glenn F Browningb, ,
Abstract;Escherichia coli is commonly isolated in canine pyometra, but little is
known of the virulence factors that may be involved in the precipitation of
this disease. The aim of this study was to compare the prevalence of
uropathogenic virulence factor (UVF) genes in E. coli isolates from canine
pyometra and from feces of healthy bitches to evaluate their role in the
pathogenesis of pyometra. E. coli from 23 cases of canine pyometra and from
the feces of 24 healthy bitches were analyzed, by polymerase chain reaction,
for UVF genes associated with canine and human urinary tract infections
(UTIs). The prevalences of UVFs in E. coli from canine pyometra were similar
to that in canine and human uropathogenic E. coli. The prevalence of pap was
greater (P=0.036) for E. coli from pyometra (52%) than for fecal isolates
(21%), and the papGIII allele was present in all pap-containing isolates. The
prevalences of genes for -haemolysin and cytotoxic necrotising factor 1
were not significantly higher (P=0.075) in E. coli from pyometra than from
feces. The proportion of pyometra strains with 3 UVFs was higher (P=0.039)
than that of fecal strains, suggesting that possession of 3 UVF genes
enhances the pathogenicity of the strain. Our findings demonstrate that E.
coli associated with canine pyometra are similar to uropathogenic strains,
and that operons that encode P fimbriae, -haemolysin and cytotoxic
necrotising factor 1 probably enhance the virulence and pathogenicity of the
strain in the canine genital tract.