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RETROSPECTIVE STUDIES
Variation of Proteinuria in Dogs with Leishmaniasis
Treated with Meglumine Antimoniate and
Allopurinol: A Retrospective Study
Marco Pierantozzi, PhD, DVM*, Xavier Roura, PhD, DVM, DECVIM-CA, Saverio Paltrinieri, PhD, DVM, DECVCP,
Marco Poggi, DVM, Andrea Zatelli, DVM
y
ABSTRACT
A retrospective study was performed using 53 client owned dogs with leishmaniasis to determine whether the degree of
proteinuria, evaluated by the urine protein/creatinine ratio (UP/C), changes following treatment with meglumine antimoniate
and allopurinol. Medical records of dogs with leishmaniasis in clinical stage C (according to the Canine Leishmaniasis Working
Group staging system) and either proteinuric or borderline proteinuric (according to the International Renal Interest Society
[IRIS] staging system) were reviewed. All dogs were treated with meglumine antimoniate and allopurinol for 4–8 wk. After
treatment, UP/C, total protein, and total globulin significantly decreased and albumin and the albumin/globulin ratio (A/G)
increased. After treatment, 7 of the 53 dogs (13.4%) became nonproteinuric following either a proteinuric or borderline
proteinuric stage. Moreover, 12 of the 53 proteinuric dogs (22.6%) changed their stage to borderline proteinuric. The anti-
leishmaniasis treatment with meglumine antimoniate in combination with allopurinol in dogs significantly reduced the degree
of proteinuria in a short period of time. The results of the current study may be useful to the veterinary practitioner in the
clinical management of canine leishmaniasis (CanL) in dogs with proteinuric chronic kidney disease. (J Am Anim Hosp Assoc
2013; 49:231–236. DOI 10.5326/JAAHA-MS-5840)
Introduction
Canine leishmaniasis (CanL) is a zoonotic parasitic disease
(enzootic in the Mediterranean basin) caused by the intracellular
protozoan Leishmania infantum. The disease is characterized
by a heterogeneous clinical presentation in which renal patho-
logic conditions are often the principal cause of death. CanL-
associated nephropathy is mainly characterized by glomerular
damage and is primarily attributed to intraglomerular deposition
of circulating immune complexes formed by organism antigens
and the antibodies produced against it.
1–3
Glomerular lesions
have been histologically classified as membranous glomerulo-
nephritis, membranoproliferative glomerulonephritis, mesangial
glomerulonephritis, and focal segmental glomerulosclerosis.
4,5
Glomerulopathy that characterizes renal involvement in CanL
causes proteinuria and renal failure. Results of previously pub-
lished studies carried out on dogs in the USA with naturally
occurring chronic renal failure suggest that persistent protein-
uria is associated with greater frequency of renal morbidity and
mortality due to both renal and extrarenal causes.
6,7
Moreover,
the risk of developing adverse outcomes increases as the mag-
nitude of proteinuria increases.
7
Nevertheless, regardless of the
role of proteinuria as a mediator of renal injury, it is an im-
portant marker both for increased risk of adverse outcomes and
for response to renoprotective interventions.
6
In a recent study,
From the Clinica Veterinaria Pirani, Reggio Emilia, Italy (M. Pierantozzi,
A.Z.); Hospital Clínic Veterinari of the Universitat Autònoma de
Barcelona, Spain (X.R.); Department of Veterinary Pathology, Hy-
giene and Public Health, University of Milan, Italy (S.P.); and Centro
Veterinario Imperiese, Imperia, Italy (M. Poggi).
Correspondence: pierantozzimarco@gmail.com (M.P.)
A/G albumin/globulin ratio; CanL canine leishmaniasis; IRIS International
Renal Interest Society; UP/C urine protein/creatinine ratio
*M. Pierantozzi’s present affiliation is Libero Professionista, Martinsicuro (TE),
Italy.
†
A. Zatelli’s present affiliation is Ospedale Veterinario I Portoni Rossi, Zola
Predosa (BO), Italy; Medical Consultancy Services, Ta’Xbiex, Malta.
ª2013 by American Animal Hospital Association JAAHA.ORG 231
allopurinol appeared to decrease proteinuria and prevent
progression of renal disease in dogs with leishmaniasis and
glomerulonephritis.
8
The aim of the current study was to investigate whether the
degree of proteinuria evaluated by the urine protein/creatinine
ratio (UP/C) in dogs with leishmaniasis classified as clinical
stage C (sick) according to the Canine Leishmaniasis Working
Group staging system changes during antiprotozoan treatment
with meglumine antimoniate in combination with allopurinol.
9
In addition, when available, the variations and correlations be-
tween serum levels of albumin, total globulin, total protein, and
albumin/globulin ratio (A/G), and respectively between all these
parameters and UP/C, both before and after treatment, were
evaluated.
Materials and Methods
Case Selection
A retrospective, multi-institutional study was performed. The
medical records of the Clinica Veterinaria Pirani (Italy), Hospital
Clínic Veterinari of the Universitat Autònoma de Barcelona
(Spain), and Centro Veterinario Imperiese (Italy) from 2006 to
2010 were reviewed to identify either proteinuric or borderline
proteinuric dogs with leishmaniasis regardless of their azotemic
status (International Renal Interest Society [IRIS] stage 1–4) and
treated with meglumine antimoniate
a,b
(75–100 mg/kg subcu-
taneously either q24 hr or divided q12 hr) in combination with
allopurinol
c,d
(20 mg/kg per os either q24 hr or divided q12 hr)
for 4–8 wk. Dogs were eligible for inclusion if they fulfilled the
following criteria:
1. A diagnosis of leishmaniasis in clinical stage C based on the
Canine Leishmaniasis Working Group staging system. Fea-
tures of clinical stage C (meaning clinically evident disease),
included dogs with positive cytologic results regardless of
serologic results, dogs with high antibody titers against
Leishmania spp., and rarely, infected dogs and one or more
clinical signs common to leishmaniasis were present. Given
the varied clinical manifestations of the disease, observed
signs suggestive of disease could differ from common clinical
signs as long as they were clearly associated with ongoing
infection. When physical examination did not reveal clinical
signs, dogs affected could still be defined as sick when he-
matologic, biochemical, and urinary alterations common to
leishmaniasis were detected. Laboratory changes other than
those considered common could also be indicative of disease,
provided that they were associated with the infection.
9
2. Negative serology for Ehrlichia canis and a negative antigen
test for Dirofilaria immitis. Moreover, dogs from an endemic
area for dirofilariasis must have been subjected to regular
prophylaxis.
3. Complete urinalysis, with inactive sediment and determina-
tion of the UP/C just before and after antileishmaniasis treat-
ment. Proteinuric before treatment was defined as a UP/C .
0.5 and borderline proteinuric was defined as a UP/C ranging
from 0.2 to 0.5 according to the IRIS staging system.
10
4. Either a deltamethrin antiparasitic collar
e,f
or permethrin-
imidacloprid spot-on dermal topical formulation
g,h
were ap-
plied during the sand fly activity period to avoid reinfection.
5. All dogs included in the study were fed a balanced commer-
cial maintenance diet.
6. Dogs must not have been treated with angiotensin-converting
enzyme inhibitors and/or an angiotensin receptor blocker in
the 2 mo period prior to and throughout the entire treatment
period.
All dogs with clinical signs and clinicopathologic findings
consistent with concurrent neoplastic, inflammatory, endocrine,
immunologic, and genetic (inherited nephropathies, either known
or suspected) diseases potentially correlated with proteinuria were
excluded. Furthermore, dogs were also excluded if they were sub-
jected to concurrent treatment with antibiotics, anti-inflammatory,
and cardiovascular drugs.
Medical Records Review
Information extracted from the medical records of each dog with
leishmaniasis included in the study consisted of signalment (age,
sex, breed), serum concentrations of creatinine, UP/C, and if
available, serum concentration of albumin, total globulin, total
protein, and A/G, just before and after treatment. Moreover, in-
formation about the length (4–8 wk) of therapy was also taken
into account. Information about the history, physical examina-
tion, complete blood count, serum biochemical analysis, complete
urinalysis, urine culture, endocrine testing, and diagnostic imag-
ing to evaluate the exclusion criteria was also collected.
All dogs recruited for this study had a diagnosis of leishmaniasis
established by clinicopathologic abnormalities, positive serology for
Leishmania infantum, and/or detection of Leishmania amastigotes
in either lymph node or bone marrow aspiration smears, or de-
tection of parasite DNA using polymerase chain reaction.
The length of antileishmaniasis treatment with the combi-
nation meglumine antimoniate and allopurinol was decided by
attending clinicians based on the patient’s clinical response and
laboratory findings. Moreover, all dogs, after the period of treat-
ment with the meglumine antimoniate/allopurinol combina-
tion, had maintenance therapy with allopurinol alone for at least
6–12 mo.
232 JAAHA | 49:4 Jul/Aug 2013
Statistical Analysis
For each set of data (i.e., for each analyte or parameter), the dis-
tribution of data recorded before and after treatment was assessed
using descriptive statistics and the I–III interquartile interval was
determined. A Shapiro-Wilk test demonstrated that data were
not normally distributed; therefore, data recorded before and
after treatment were compared using a Wilcoxon signed rank
test. Either pretreatment or posttreatment concentrations of serum
total protein, albumin, total globulin, and UP/C were correlated to
each other, and degree and significance of the correlations were
determined using the Spearman rank correlation (r). The level of
significance was set at P,0.05, and all calculations were per-
formed using a commercial software program.
i
Results
The medical records of 280 dogs with leishmaniasis were reviewed,
and 53 cases met inclusion criteria. The remaining 227 dogs were
excluded because of concurrent treatment with antiproteinuric
medications, such as renal diet and angiotensin-converting enzyme
inhibitors, and/or angiotensin receptor blocker (n ¼141), anti-
biotics (amoxicillin trihydrate/clavulanate potassium, cefalexin,
enrofloxacin, metronidazole, or doxycycline (n ¼9), cardiovas-
cular drugs such as furosemide, spironolactone, angiotensin-
converting enzyme inhibitors, pimobendan, or amlodipine (n ¼
11), anti-inflammatory drugs such as glucocorticoids or nonste-
roidal anti-inflammatory drugs (n ¼6), diagnosis of leishmaniasis
not in clinical stage C (n ¼28), positive serology for Ehrlichia
canis (n ¼9), insulinoma (n ¼1), hyperadrenocorticism (n ¼1),
incomplete diagnostic data pre- and postantileishmaniasis treat-
ment (n ¼12), suspected splenic neoplasia (n ¼1), suspected
perianal adenoma (n ¼1), suspected inherited nephropathies
(n ¼2), and lack of preventive measures to avoid reinfection
(n ¼5). Of the 53 cases enrolled, 34 dogs were purebreeds
(belonging to 18 breeds) and 19 were mixed-breeds. Thirty-two
dogs (60.4%) were males (3 castrated) and 21 dogs (39.6%) were
females (7 spayed). Median age was 6 yr (mean, 6.05 yr; range,
2–17 yr). Forty-one dogs (77.4%) were treated for 4 wk, 4 dogs
(7.5%) for 5 wk, 5 dogs (9.4%) for 6 wk, and 3 dogs (5.7%) for
8 wk. The median duration of therapy was 4 wk (mean, 4.49 wk;
range, 4–8wk).
Results regarding serum protein concentration and UP/C
ratio have been summarized in Table 1. A significant reduction
of the concentration of total protein, total globulin, and UP/C was
seen after treatment compared with pretreatment levels, as well as
a significant increase of the concentration of albumin and the
A/G. In some cases, however, despite the trend to normalization,
mean and median posttreatment values remained outside the
reference ranges.
Before treatment, a significant negative correlation was found
between UP/C and both serum albumin concentration (r¼
20.41; P¼0.029) and A/G (r¼20.39; P¼0.006), and a sig-
nificant positive correlation was found between UP/C and total
globulin (r¼0.31; P¼0.027). No significant correlation between
UP/C and total protein was found (r¼0.27; P¼0.062). After
treatment, no significant correlations were observed between
UP/C and total protein (P¼0.102), albumin (P¼0.531), total
globulin (P¼0.083), or A/G (P¼0.297).
Creatinine values were used only for the IRIS staging system
before and after antileishmaniasis treatment. Before treatment, 40
of 53 dogs (75.5%) were IRIS stage 1 and proteinuric, 11 of 53 dogs
(20.7%) were IRIS stage 1 and borderline proteinuric, and 2 of
53 dogs (3.8%) were IRIS stage 2 and proteinuric. After treat-
ment, 27 of 3 dogs (51%) were IRIS stage 1 and proteinuric, 18
of 53 dogs (34%) were IRIS stage 1 and borderline proteinuric,
7 of 53 dogs (13.1%) were IRIS stage 1 and nonproteinuric, and
1 of 53 dogs (1.9%) was IRIS stage 3 and proteinuric (Table 2).
After treatment, 7 of 53 dogs (13.2%) went from either a
proteinuric or borderline proteinuric stage to a nonproteinuric
stage, and 12 of 53 proteinuric dogs (22.6%) changed their stage
from proteinuric to borderline proteinuric (Table 3). Moreover,
TABLE 1
Results Recorded Before and After Antileishmaniasis Treatment in 50 Paired Samples*
Pretreatment Posttreatment Pvalue
y
Reference range
Total protein (g/L) 80.0 (67.0–91.3) 68.0 (62.7–77.3) ,0.001 52–71
Albumin (g/L) 21.6 (17.2–25.0) 26.0 (21.7–30.1) ,0.001 27–38
Total globulin (g/L) 57.0 (48.5–69.1) 42.9 (37.7–49.3) ,0.001 25–45
A/G 0.37 (0.28–0.50) 0.60 (0.46–0.75) ,0.001 0.5–1.3
UP/C
x
1.02 (0.56–2.13) 0.55 (0.31–1.07) ,0.001 0.0–0.5
* Data are reported as median (I–III interquartile interval).
y
The Pvalue indicates the level of statistical significance between pre- and posttreatment results (Wilcoxon signed rank test).
x
UP/C values are based on 53 paired samples.
A/G, albumin/globulin ratio; UP/C, urine protein/creatinine ratio.
Proteinuria Pre- and Postantileishmaniasis Treatment in Dogs
JAAHA.ORG 233
24 of 53 dogs (45.3%) were proteinuric before and after treatment,
albeit with a significant reduction in the degree of proteinuria. Only
4 of 53 dogs (7.5%) remained proteinuric with a slight increase in
the degree of proteinuria. Six of 53 dogs (11.3%) were borderline
proteinuric before and after treatment. After treatment, 13 dogs
(24.5%) had modified their need for antiproteinuric therapy
according to the IRIS treatment recommendations based on val-
ues of serum creatinine and UP/C (Table 4).
Discussion
Proteinuric chronic kidney disease is frequently found in dogs
with leishmaniasis. In CanL, glomerular deposition of circulating
immune antigen/antibody complexes plays an essential role in the
pathogenesis of renal injury, with subsequent development of
proteinuria.
1–5
There is strong evidence that proteinuria is a risk
factor for the development and progression of renal failure, and
that antiproteinuric treatment with dietary modification and
angiotensin-converting enzyme inhibitors reduces protein excre-
tion and disease progression in dogs with glomerulonephritis.
11
The correct therapeutic management of dogs affected by
naturally occurring proteinuric glomerulonephritis involves identify
and treating any potential underlying disease processes, including
reduction of proteinuria and management of uremia and other
complications of generalized renal failure.
12
In the current study, the authors included all patients treated
with the most widely used and recommended treatment protocol
for dogs with leishmaniasis: the combination of meglumine anti-
moniate, which selectively inhibits leishmanial glycolysis and fatty
acid oxidation, and allopurinol, which inhibits protein translation
by interfering with RNA synthesis.
13–15
In the current study, laboratory findings detected before
treatment (i.e., hyperproteinemia, hypoalbuminemia, hyper-
globulinemia, decreased A/G, proteinuria) were consistent with
those previously reported by other authors.
16,17
The hyper-
proteinemia caused by hyperglobulinemia observed in this study
is in agreement with the findings of other authors who reported
that in some dogs with leishmaniasis there is an increase in total
protein levels due to a greater production of antibodies.
16
With
regard to serum albumin, the renal loss, a decreased synthesis
consistent with the role of albumin as a negative acute phase
protein, and decrease protein intake may represent the most likely
cause of hypoalbuminemia in CanL.
18
In this study, the authors
found a significant negative correlation between UP/C and serum
albumin concentration before treatment, as previously reported.
19
After treatment, an increase in albumin values associated with
a reduction of the UP/C, although the correlation between those
two parameters was not statistically significant. Considering that
all dogs included in this study were fed a balanced diet without
TABLE 2
Number of Dogs Classified According to the IRIS Staging System Pre- and Posttreatment for Leishmaniasis
Pretreatment Posttreatment
IRIS stage Nonproteinuric Borderline proteinuric Proteinuric Nonproteinuric Borderline proteinuric Proteinuric
1 0 11 40 7 18 27
20 020 00
30 000 01
40 000 00
IRIS, International Renal Interest Society.
TABLE 3
UP/C Values of 19 Samples that Changed the IRIS Substage of
Proteinuria Pre- and Postantileishmaniasis Treatment
UP/C
pretreatment Interpretation
UP/C
posttreatment Interpretation
0.30 Borderline proteinuric 0.10 Nonproteinuric
0.30 Borderline proteinuric 0.10 Nonproteinuric
0.40 Borderline proteinuric 0.13 Nonproteinuric
0.40 Borderline proteinuric 0.10 Nonproteinuric
0.45 Borderline proteinuric 0.09 Nonproteinuric
0.53 Proteinuric 0.15 Nonproteinuric
0.85 Proteinuric 0.00 Nonproteinuric
0.53 Proteinuric 0.28 Borderline proteinuric
0.58 Proteinuric 0.49 Borderline proteinuric
0.68 Proteinuric 0.28 Borderline proteinuric
0.68 Proteinuric 0.38 Borderline proteinuric
0.70 Proteinuric 0.35 Borderline proteinuric
0.80 Proteinuric 0.49 Borderline proteinuric
0.89 Proteinuric 0.43 Borderline proteinuric
0.90 Proteinuric 0.32 Borderline proteinuric
1.10 Proteinuric 0.45 Borderline proteinuric
1.18 Proteinuric 0.49 Borderline proteinuric
1.87 Proteinuric 0.38 Borderline proteinuric
1.98 Proteinuric 0.46 Borderline proteinuric
IRIS, International Renal Interest Society; UP/C, urine protein/creatinine ratio.
234 JAAHA | 49:4 Jul/Aug 2013
a reduction in daily caloric intake throughout the treatment pe-
riod, it is likely that the hypoalbuminemia might have been due
mainly to the renal loss.
Similar to a recent study, antileishmaniasis treatment reduced
the magnitude of proteinuria.
8
Nevertheless, in contrast to the
aforementioned study, which showed a significant reduction (P¼
0.0005) in proteinuria after 6 mo of administration of allopurinol
(10 mg/kg q12 hr), the results reported herein showed a statisti-
cally significant reduction of UP/C after 4–8 wk of treatment.
One possible explanation may be related to the mechanism of
action of the two drugs used. Although allopurinol acts only as
a leishmaniostatic drug, meglumine antimoniate is considered
leishmanicidal and also increases the phagocytic capacity of
monocytes and neutrophils.
20–23
Thus, the combined use of
meglumine antimoniate and allopurinol could lead to a more rapid
reduction of parasitic load, and consequently, of the circulating
immune complexes that affect the kidneys. That hypothesis is
supported by the results of previous studies on the follow-up of
dogs with leishmaniasis treated with meglumine antimoniate
and allopurinol that showed a marked decrease of parasitic
load.
24,25
Furthermore, several studies demonstrated that the
rapid clinical efficacy of meglumine antimoniate with allopuri-
nol mostly depends on meglumine antimoniate, rather than on
allopurinol, as demonstrated by trials in which the efficacy of
meglumine antimoniate and allopurinol was similar to those
already described for meglumine antimoniate alone.
20
Such a rapid reduction of proteinuria was also observed in
human visceral leishmaniasis (Leishmania donovani) when a
10 day treatment with N-methyl-glucamine was applied.
26
In the current study, the authors observed a statistically
significant reduction in the grade of proteinuria posttreatment.
In addition, 13 of 53 dogs (24.5%) modified their creatinine
and UP/C values; thus, according to the IRIS treatment rec-
ommendations, they did not need antiproteinur ic therapy
(i.e., angiotensin-converting enzyme inhibitors and renal diet)
(Table 4).
10
The results of this study provides an important
and clinically relevant finding that may be useful in the clinical
management of proteinuric dogs with leishmaniasis in clinical
stage C. In addition, this finding may have some clinical use
when planning an antiproteinuric therapy concurrent to the
antileishmaniasis treatment with the meglumine antimoniate/
allopurinol combination and in evaluating its therapeutic
efficacy.
There were several limitations to the current study, with the
most important being its retrospective nature. Although the
majority of dogs included were treated for 4 wk, the lack of a
standardized treatment protocol for the length of therapy was
another important limitation. Moreover, due to lack of data, it was
not possible to evaluate blood pressure in relation to other lab-
oratory parameters (i.e., UP/C, albumin, total globulin, total
protein, A/G) for the purpose of IRIS staging.
Conclusion
To the best of the authors’knowledge, this is the first study re-
porting a statistically significant reduction in the magnitude of
proteinuria in dogs with leishmaniasis in clinical stage C sub-
mitted only to antileishmaniasis treatment with the combination
meglumine antimoniate and allopurinol. This information may
TABLE 4
Results of the 13 Dogs that Changed Their Need for Antiproteinuric Therapy According to the IRIS Treatment Recommendations Based
on Values of Serum Creatinine and UP/C
IRIS stage pretreatment IRIS stage posttreatment UP/C pretreatment UP/C posttreatment Tx pretreatment Tx posttreatment
1 1 6.1 0.7 Yes No
1 1 2.66 1.48 Yes No
1 1 5.9 0.55 Yes No
1 1 4.9 0.88 Yes No
1 1 4.49 1.3 Yes No
1 1 2.6 1.3 Yes No
1 1 2.1 0.8 Yes No
1 1 4.29 1.35 Yes No
1 1 2.2 0.8 Yes No
1 1 3.3 1.2 Yes No
2 1 0.98 0.6 Yes No
2 1 1.1 0.45 Yes No
2 2 1.18 0.49 Yes No
IRIS, International Renal Interest Society; Tx, antiproteinuric therapy; UP/C, urine protein/creatinine ratio.
Proteinuria Pre- and Postantileishmaniasis Treatment in Dogs
JAAHA.ORG 235
be useful to the veterinary practitioner in the management of
proteinuria in dogs with leishmaniasis.
FOOTNOTES
a
Glucantime; Merial, Milano, Italy
b
Glucantime; Merial, Barcelona, Spain
c
Zyloric; GlaxoSmithKline, Teofarma s.r.l., Pavia, Italy
d
Zyloric; Faes Farma S.A., Leioa, Spain
e
Scalibor Protector Band; Intervet, Milano, Italy
f
Scalibor Protector Band; Intervet, Madrid, Spain
g
Advantix; Bayer, Milano, Italy
h
Advantix, Bayer, Barcelona, Spain
i
Analyse-it version 2.26; Analyse-it Software Ltd., Leeds, United
Kingdom
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