Early decrease of ionized calcium and static symmetric

American Journal of Veterinary Research 1

enal lesions have been reported in 40% of cap-

tive chameleons

and 64% of Hermann tortoises

(Testudo hermanni)

at necropsy. As renal disease is

frequent in reptiles, there is a need to develop nonin-

vasive diagnostic tests to detect early stages of dis-

ease. In snakes, collection of uncontaminated urine

is challenging as they lack a urinary bladder, making

Early decrease of ionized calcium and static symmetric

dimethylarginine concentration in a model of renal

tubular necrosis in corn snakes (Pantherophis guttatus)

Claire Vergneau-Grosset, DMV, IPSAV, CES, DACZM

*; Julie Pujol, DMV, DES

; Bastien Rubin, BSc

;

Jacobo Romano Noriega, MVZ

; Carolyn Gara-Boivin, DMV, MSc, DACVP

; Younes Chor, DMV, MSc, PhD

;

Shannon Ferrell, DMV, DACZM, DABVP (Avian Practice)

; Stéphane Lair, DMV, IPSAV, DVSc, DACZM

Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada

Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada

Département de Biomédecine, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada

*Corresponding author: Dr. Vergneau-Grosset (claire.grosse[email protected])

blood parameters particularly relevant as biomarkers

of renal disease.

Other diagnostic modalities, such as glomeru-

lar ltration rate measurement and scintigraphy,

have been described in reptiles but remain mar-

ginally used in clinical practice due to their limited

availability or patient hospitalization requirements.

Glomerular ltration rate references have been pub-

lished for lizards

4,5

and turtles,

but they vary with

hydration status

and ambient temperature outside

of the preferred optimal temperature zone,

com-

plicating clinical interpretation of results. An ideal

OBJECTIVE

To determine if plasma concentrations of symmetric dimethylarginine (SDMA), N-acetyl-beta-

d-glucosaminidase

(NAG), GGT, ALT, AST, lactate, total calcium, and ionized calcium (iCa) and the calcium:phosphorus ratio are clini-

cally relevant biomarkers to detect early stages of tubular lesions in snakes.

ANIMALS

6 adult corn snakes (Pantherophis guttatus).

METHODS

Corn snakes were administered 11 injections of gentamicin at 50 mg/kg, SC, q 24 h in an experimental model of

induced tubular necrosis. Plasma biochemistry and blood gas analyses were performed at baseline and after the 3rd

and 11th injections. Parameters were compared between time points using a paired Wilcoxon test. In 3 individuals,

renal biopsies were collected at baseline before starting injections and at the 3rd and 11th injections, while renal

tissue samples were procured after euthanasia in all individuals.

RESULTS

Renal proximal and distal tubular necrosis and hepatic steatosis were present in all individuals at necropsy. Compared

to baseline, decreased blood concentrations of lactate, ionized calcium, and total calcium and a decreased

calcium:phosphorus ratio were noted. A signicant decrease of lactate and ionized calcium was observed after

3 days. Conversely, no changes in SDMA, NAG, ALT, AST, GGT, and sodium were detected.

CLINICAL RELEVANCE

Ionized calcium and lactate concentrations were the earliest parameters to decrease compared to baseline values

in this experimental model. While SDMA is a sensitive indicator of renal disease in mammals, this biomarker did not

increase in a model of induced acute tubular necrosis in corn snakes.

Keywords: kidney, NAG, N-acetyl-beta-d-glucosaminidase, SDMA, symmetric dimethylarginine

Received September 27, 2023

Accepted November 8, 2023

doi.org/10.2460/ajvr.23.09.0204

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2 AJVR

marker of renal disease in reptiles should be com-

mercially available and provide a rapid single-point

result with good sensitivity/specicity.

Symmetric dimethylarginine (SDMA) is a sen-

sitive early marker of acute and chronic renal dis-

ease in dogs and cats.

8–10

Since 2019, SDMA has

been included in the International Renal Interest

Society staging criteria in dogs and cats. It has been

detected in Hermann tortoises and proposed as a

marker of renal injury in a retrospective study involv-

ing 28 individuals with hyperuricemia.

Reference

intervals for SDMA have also been described in green

sea turtles (Chelonia mydas).

However, the clini-

cal relevance of this marker remains unclear in rep-

tiles. A recent study

has shown that GGT and ALT

activities were higher in the kidneys of healthy water

snakes (Nerodia spp) than in other tissues evaluated.

Therefore these biochemistry parameters could be of

clinical interest if they are increased when compared

to reference intervals in cases with renal lesions.

N-acetyl-beta-

d-glucosaminidase (NAG) is a lyso-

somal enzyme, which has been described as a marker

of acute tubular necrosis in birds.

14–16

It is detectable

in reptile plasma.

Among tested organs, NAG is

found preferentially in the renal tissue of pigeons,

but its tissue distribution is currently unknown in

reptiles. Evaluating the organ distribution of NAG in

snakes would be relevant to determine if this enzyme

is predominantly present in renal tissue.

The objectives of this study were twofold: rst,

to compare the sensitivity of commercially avail-

able blood markers to detect early stages of renal

disease in corn snakes (Pantherophis guttatus), and

second, to evaluate the tissue distribution of NAG in

this species. Our hypotheses were that plasma SDMA

and NAG concentrations would be more sensitive

than other biochemistry parameters to detect renal

disease in snakes and that NAG would be mainly

detected in renal tissue as opposed to other evalu-

ated organs in a healthy corn snake.

Methods

This experimental study was approved by the

Animal Care and Use Committee of the Université

de Montréal (21-Rech-2134). Seven adult corn

snakes (5 males, 2 females; body weight, 241 to

862 grams) were purchased from a breeder. Animals

were considered healthy after a complete physi-

cal examination, a clinical follow-up over 3 weeks,

and unremarkable fecal examinations (direct smear

and otation in zinc sulfate). One of the 7 snakes, a

4-year-old adult female, was kept as a control in the

same environment as the other individuals involved

in the experiment.

Snakes were kept individually in glass terraria

with a surface of 90 X 45 cm (Exoterra; Hagen Inc).

They were housed in a temperature and humidity-

controlled room set at 29 °C and 38% to 53%, respec-

tively. A warm area at 31°C was provided in each

terrarium by heat lamps. A large water bowl (Corner

bowl; Zoomed) was provided where snakes could

immerse themselves completely. The photoperiod

was set at a light:darkness ratio of 12:12. Paper was

used as substrate, and a hiding box was provided

in the colder area of each terrarium. Snakes were

weighed twice weekly. They were fed thawed mice

once a week. Prey items were oered again after

2 days in case of anorexia. Snakes were not handled

for 24 hours after each successful prey ingestion.

The 6 corn snakes were randomly divided into

2 groups: 1 group (n = 3) underwent a renal biopsy

per week, while the other group (3) only had a sin-

gle renal biopsy before starting the experiment to

conrm the absence of renal histological changes.

The goal of having 2 groups was to evaluate the

potential eects of renal biopsies on skeletal mus-

cular enzymes (ie, creatine kinase, ALT, and AST

)

independently of the eect of gentamicin. To induce

renal tubular necrosis, SC injections of gentami-

cin (100 mg/mL Gentocin; Merck Animal Health)

at 50 mg/kg

were administered in 6 corn snakes

(5 males, 1 female) in the caudo-dorsal area of the

body daily for 11 injections or until euthanasia. In

case of ecdysis, injections were discontinued for

2 days to allow for complete shedding. Limit points

used during the project were based on a grading sys-

tem established before the study (Supplementary

Table S1). Euthanasia was elected for individuals in

which a score of 3 was reached for any 1 parameter

in combination with a score ≥ 2 for another param-

eter or at 21 days as per study design. If gout was

detected during coelomic ultrasounds, which were

performed weekly (MyLab Sigma; Esoate Canada),

euthanasia was also elected.

Before the beginning of the study (time point T0)

and after a 3-week acclimation period, blood samples

were collected in each of the 7 snakes under general

anesthesia with isourane at 5% administered in 100%

oxygen. Blood collection was performed via car-

diocentesis to avoid lymph contamination and was

stored in lithium heparin tubes (Lithium Heparin BD

Microtainer Tubes; Becton, Dickinson and Company).

The procedure was repeated in 6 snakes after 3 injec-

tions (time point T3) and 11 injections of gentamicin

(time point T11). All blood samples were performed

before surgery when applicable, and after at least

a 48-hour fast. Plasma biochemistry panels were

submitted immediately to the Clinical Pathology

Service of the Centre de Diagnostic Vétérinaire de

l’Université de Montréal for each snake and included

uric acid, BUN, total calcium, phosphorus, total

proteins, GGT, ALT, ALP, AST, total cholesterol, CK,

glucose, LDH, NAG, and biliary acid concentrations

using standard techniques (Table 1). The NAG con-

centration was measured using commercial reagents

(Diazyme Laboratories), as described in birds.

Frozen plasma was shipped on ice to measure SDMA

(Idexx Laboratories). No plasma biochemistry values

published in peer-reviewed literature were available

for corn snakes. Reference values obtained from a

global database from various analyzers were used

for interpretation (number of snakes = 65 to 140

depending on parameters).

Blood pH, ionized cal-

cium (iCa), sodium, potassium, chloride, lactate, par-

tial pressure in carbon dioxide, and other standard

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AJVR 3

analyte concentrations were obtained on a handheld

blood gas analyzer at 31 °C (Heska Element POC;

Epocal Inc). For pH and iCa, temperature-adjusted

values were calculated as previously described in

snakes.

Blood concentrations of BUN were mea-

sured on both the biochemistry panel and blood

gas analysis. Since the detection limit for BUN was

3 mg/dL on the blood gas analysis and 0.3 mg/dL on

the laboratory analyzer, plasma BUN concentrations

measured by the laboratory analyzer were used for

statistical analyses. Reference values using the same

blood gas analyzer for free-ranging snakes of the

genus Pantherophis from a previous study (number

of snakes = 19)

and reference values for iCa pub-

lished in ball pythons (Python regius)

exposed to

articial ultraviolet light were used for interpretation.

Surgical renal biopsies were performed under

general anesthesia before the beginning of the study

in all snakes and after 3 and 11 gentamicin injections

in 3 of the 6 snakes. Premedication included 0.5 mg/

kg hydromorphone (2 mg/mL HYDROmorphone;

Sandoz Canada Inc) SC administered 1 hour before

induction and 5 mg/kg alfaxalone, SC (10 mg/ml

Alfaxan Multidose; Jurox Pty Ltée) both in the cranial

third of the body, although no signicant dierence

in anesthesia duration or physiological variables has

been reported in corn snakes among injection sites

for alfaxalone.

Snakes were induced by mask with

5% isourane (USP; Fresenius Kabi Canada Ltd) in

100% oxygen and intubated with uncued endo-

tracheal tubes constructed from a 16-G catheter

(Terumo Medical Canada Inc). Snakes were manu-

ally ventilated twice per minute. Monitoring included

a Doppler ow detector (Ultrasonic Doppler Flow

Detector; model 811-B; Parks Medical Electronics)

placed over the heart and a capnometer (Covidien

N-85; Nellcor). All surgeries were performed in

a temperature-controlled room set at 29 °C. The

caudal part of the snake was placed in right lateral

recumbency. The left kidney was located at a dis-

tance of 90% of the head to cloacal length and visual-

ized by ultrasound. Then, 4 mg/kg lidocaine (20 mg/

ml Lurocaine; Vetoquinol) was injected SC at the

incision site. The left kidney was approached in a

standard fashion. Two mosquito hemostatic forceps

were placed laterally on the kidney for 2 minutes

before obtaining a 3-mm wedge biopsy. Hemostasis

was veried, and the biopsy was placed in a cassette

in buered 10% formalin. If hemostasis was partial,

a 5-mm section of procoagulant absorbable gelatin

sponge (Surgifoam; Ethicon Inc) was placed on the

biopsy site. The coelomic muscles were closed with

a continuous pattern using polydioxanone suture

(PDS 4.0; Ethicon; Johnson and Johnson Medtech).

The skin was closed with interrupted sutures in an

everting pattern using PDS 4.0. An injection of 2 mg/

kg ketoprofen (100 mg/ml Anafen; Merial Canada)

was administered in the cranial epaxial muscles.

Euthanasia was performed under isourane

anesthesia with an intracardiac injection of 100 mg/

kg pentobarbital sodium (340 mg/ml Euthanyl;

Bimeda-MTC Animal Health) and 2 mEq/kg potas-

sium chloride (2 mEq/ml; Pzer Canada) followed

by brain pithing. A complete necropsy was then per-

formed, and tissue samples were taken and xed in

buered 10% formalin, including the kidneys, heart,

lung, liver, spleen, stomach, and reproductive tract.

Four sections along the length of the right kidney

were obtained and examined. Tissues were stained

with hematoxylin, phloxine, and saron using a rou-

tine staining protocol. Histopathologic scoring of

kidney sections (biopsies and necropsy specimens)

was performed, without prior knowledge of individ-

uals and time points by a single veterinary pathol-

ogy resident (SF) under the supervision of a trained

and experienced veterinary pathologist (SL). A total

of 5 randomly selected, renal elds at a magnica-

tion of X200 were evaluated from each biopsy and/

or necropsy submission. Each eld was scored using

a standardized semiquantitative scale (0, within

normal limits/absent; 1, mild changes [< 10% of the

cells or eld aected]; 2, moderate changes [10% to

50%]; and 3, marked changes [> 50%]). Parameters

scored were (1) proximal tubule degeneration,

inammation, and necrosis; (2) distal tubule degen-

eration, inammation, and necrosis; (3) severity of

intraluminal mineral deposition; and (4) intraluminal

urate deposition. Tubular degeneration was charac-

terized by pale, swollen, vacuolated epithelial cells

with a loss of cytoplasmic detail, while necrosis was

dened by cytoplasmic hypereosinophilia, a loss of

cellular borders, and nuclear pyknosis/karyorrhexis.

The median value of the 5 evaluated elds was then

reported as the scored result for each parameter for

the specimen.

Sections of liver from each animal harvested at

necropsy were processed and stained as described

above to check for evidence of hepatic degen-

eration/necrosis. A total of 5, randomly selected

hepatic elds (magnication, X200) were evalu-

ated and scored with a standardized semiquan-

titative scale: (0, within normal limits/absent; 1,

Table 1—Analytic methods used to measure plasma

concentration of metabolites in corn snakes (Pantherophis

guttatus) using a laboratory analyzer (Beckman Coulter

DxC 600)

Parameter Analytical method

Glucose Hexokinase, 340 nm

Urea Enzymatic, 340 nm

ALT Henry, 340 nm

AST Henry, 340 nm

ALP Kinetic rate, 410 nm

CK Rosalki, 340 nm

GGT Szasz, 410 nm

LDH Lactate to pyruvate, 340 nm

Total calcium Endpoint, indirect ion selective

electrode, calcium ionophore

membrane

Phosphorus Phospho-molybdate, 340 nm

Total cholesterol Enzymatic, 520 nm

Total proteins Biuret, 560 nm

Bile acids Enzymatic, 405 nm

NAG Colorimetric, 505 nm

CK = Creatinine kinase. NAG = N-acetyl-beta-

glucosaminidase.

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4 AJVR

mild changes [< 10% of the cells or eld aected];

2, moderate changes [10% to 50%]; and 3, marked

changes [> 50%]). Parameters scored were (1) ste-

atosis, (2) inammation, (3) necrosis, (4) gout tophi,

and (5) brosis. The median of the scored result was

reported for each parameter for the specimen.

In addition to the experimental study, an adult

female corn snake was kept in the same environmen-

tal conditions and was euthanized with the same

protocol. A normal health status was conrmed by

necropsy and a plasma biochemistry panel within

reference ranges.

A wet weight of 0.58 grams from

each organ (kidney, liver, heart, pancreas, small intes-

tine, ovary, and skeletal muscle) was sampled using

a scale with a precision of 0.1 mg (Sartorius Canada

Inc). Each tissue was homogenized for 10 minutes

in a microtube with glass beads and 1 mL of ultra-

pure water (Mini-Beadbeater 96; Biospec Products

Inc). The content was centrifuged at 15,060 X g for

10 minutes (Centrifuge 5425; Eppendorf Canada

Ltd), and the supernatant was submitted for NAG

concentration measurement. The same NAG com-

mercial assay kit was adapted to run on an auto-

mated chemistry analyzer (Beckman Coulter).

Data were analyzed using a statistical analysis

software (R version 4.2.1, R Core Team [2022]. R:

A language and environment for statistical comput-

ing. R Foundation for Statistical Computing. https://

www.R-project.org/). For each blood parameter, the

initial value measured on each individual and the

values obtained after 3 and 11 injections of genta-

micin (time points called T3 and T11 for all snakes)

were compared using paired Wilcoxon tests. When a

parameter had a value below the detection limit of

a test, the lowest measurable value of the test was

used for statistical analysis. A Benjamini-Hochberg

correction was applied to the P values. To evalu-

ate the correlation of biochemical parameters with

proximal and distal tubular necrosis lesions, a linear

mixed model was created with each blood parameter

as the dependent variable (including uric acid, lac-

tate, sodium, total calcium, iCa, calcium:phosphorus

ratio, ALT, AST, and urea), the histologic score as an

independent variable, and the snake specimen as

a random variable. The R package ‘lme4’ was used

for this analysis. The signicance of the variable was

tested via a likelihood ratio test. Data distribution of

the residues of each model was evaluated using a

Shapiro-Wilk normality test. A logarithmic transfor-

mation of the ALT variable was used to achieve nor-

mality of the model residues. The level of statistical

signicance was set at .05.

Results

Baseline blood gas analysis results were

within published reference ranges

19,21

except for

1 sample with slightly decreased lactate concen-

tration (1.69 mmol/L; reference values, 2.66 to

20.1 mmol/L).

Baseline renal histology was within

normal limits in all 6 snakes involved in the experi-

mental study, including 2 snakes with mild renal

changes considered incidental and insucient

for exclusion from the study. During the gentami-

cin injections, none of the snakes developed renal

lesions that were detectable on coelomic ultrasound.

Snakes reached limit points at the 11th injection of

gentamicin which justied euthanasia at that time.

Clinical signs developing during the second week

of the study varied by individual and included diar-

rhea, polydipsia, polyuria, lethargy, and anorexia.

Observed renal pathologic changes are summarized

(Figure 1). Since all renal biopsies performed at T11

for the 3 snakes had identical scores as the results

obtained at necropsy, renal scores at necropsy are

displayed for these 3 snakes. As the injections pro-

gressed, the proximal tubules displayed a mild to

moderate degeneration with more frequent, intense

levels of necrosis, while the distal tubules more fre-

quently demonstrated higher levels of degeneration

with less frequent, intense necrosis (Table 2). Little

to no inammation was associated with the afore-

mentioned tubular degeneration/necrosis in either

the proximal or distal segments. Mild to moderate

intratubular mineralization was observed in both

baseline (T = 0) and biopsy/necropsy samples with

no obvious increase in frequency or intensity as the

study progressed. Intratubular luminal urate deposi-

tion was not observed in any submitted sample at

any time point. On necropsy, hepatic steatosis was

noted in all snakes, and 1 snake displayed multiple

hepatic tophi characterized by foci of urate crystal

deposition, hepatocellular necrosis, and a moderate

to marked heterophilic inltrate with macrophages.

Evolution of the plasma biochemistry panel

parameters is illustrated (Figure 2). Uric acid con-

centration did not increase signicantly between

T0 and T3 (P = .22), although there was a tendency

toward an increase between T0 and T11 (P = .06)

and 4 out of 6 snakes displayed hyperuricemia at T11

(reference, 168 to 1,194 µmol/L

Plasma concentrations of ALP, BUN, CK, LDH,

and total proteins did not change signicantly over

time and remained within reference intervals for corn

snakes.

Plasma CK concentrations remained similar

in both groups. No signicant dierences in SDMA,

NAG, AST, ALT, and GGT concentrations were found

between baseline and any time point. All measured

AST concentrations remained within reference inter-

vals

at all time points except for 2 samples at T11.

Total calcium concentrations and the

calcium:phosphorus ratio decreased signicantly

between T0 and T3 (P = .03) and between T0 and

T11 (P = .03). Two out of 6 snakes had a slightly

decreased total calcium at T3 (range, 3.16 to

3.29 mmol/L; reference, 3.38 to 4.90 mmol/L). No

signicant changes were noted in plasma phospho-

rus concentrations overtime. All 6 snakes displayed

calcium:phosphorus ratios within reference values

at T3.

Four out of 6 snakes displayed a decreased

calcium:phosphorus ratio at T11 (range, 1.4 to 2.1;

reference, 2.2 to 9.7).

On blood gas analysis, iCa decreased signicantly

between T0 and T3 (P = .03) and between T0 and

T11 (P = .03) (Figure 3). All snakes had baseline iCa

concentrations within the published reference range

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AJVR 5

for ball pythons (1.67 to 1.91 mmol/L; n = 14).

iCa

concentrations fell below reference values in 5 out

of 6 snakes at T3 (range, 1.39 to 1.64 mmol/L) and

in all 6 snakes at T11 (range, 1.02 to 1.46). Lactate

concentration decreased signicantly between T0

and T3 (P = .03) and between T0 and T11 (P = .03).

Figure 1—Histogram of individual median scores of tubular lesions in 6 corn snakes (Pantherophis guttatus) under-

going renal biopsies before the beginning of the study (T0) and in 3 corn snakes after 3 injections (T3) and after

11 injections (T11) of gentamicin 50 mg/kg, SC, in the caudal third of the body (renal biopsy or necropsy results).

Table 2—Histologic median score values regarding renal lesions in 6 corn snakes (P guttatus) undergoing renal

biopsies before the beginning of the study (T0), in 3 corn snakes after 3 injections (T3) and after 11 injections (T11)

of gentamicin, and in all 6 corn snakes at necropsy.

Histology median score values

Snake ID Sample PTD PTI PTN DTD DTI DTN ITM IUD

1 BIOPSY 1 0 0 0 0 0 0 0 0

1 NECROPSY 0 0 3 1 0 1 0 0

2 BIOPSY 1 0 0 0 0 0 0 0 0

2 BIOPSY 2 2 0 1 3 0 0 1 0

2 BIOPSY 3 0 0 3 2 0 1 0 0

2 NECROPSY 0 0 3 2 0 1 0 0

3 BIOPSY 1 0 0 0 0 0 0 0 0

3 NECROPSY 0 0 3 1 0 1 1 0

4 BIOPSY 1 0 0 0 0 0 0 0 0

4 NECROPSY 3 0 2 2 0 1 0 0

5 BIOPSY 1 2 0 0 2 0 0 0 0

5 BIOPSY 2 2 0 0 2 0 1 0 0

5 BIOPSY 3 0 0 3 2 0 1 0 0

5 NECROPSY 0 0 3 3 0 2 0 0

6 BIOPSY 1 2 0 0 1 0 0 2 0

6 BIOPSY 2 2 0 1 2 0 0 1 0

6 BIOPSY 3 0 1 3 3 0 1 1 0

6 NECROPSY 0 1 3 2 1 0 1 0

DTD = Distal tubule degeneration. DTI = Distal tubule inammation. DTN = Distal tubule necrosis. ITM = Intratubular mineral

deposition. IUD = Intraluminal urate deposition. PTD = Proximal tubule degeneration. PTI = Proximal tubule inammation.

PTN = Proximal tubule necrosis.

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6 AJVR

At T3, 4 out of 6 snakes displayed lactate concen-

trations below reference values.

At T11, lactate

concentrations fell below the limit of detection

of 0.3 mmol/L in 5 out of 6 snakes. No signicant

changes were observed among time points for blood

pH (P = .31) or partial pressure of carbon dioxide

(P = .09). No signicant changes were noted for

potassium, sodium, and chloride concentrations. At

the third time point (T11), sodium was below the

reference values in 4 out of 6 snakes (range, 141 to

148 mmol/L; reference, 153 to 180 mmol/L).

Proximal tubular necrosis was signicantly

correlated with uric acid (P < .001), total calcium

(P < .001), iCa (P < .001), lactate (P < .001), ALT (P <

.001), BUN (P < .001), sodium (P < .001), and AST

(P = .008) concentrations and calcium:phosphorus

ratio (P = .006). Distal tubular necrosis was signi-

cantly correlated with decreased total calcium (P =

.002), decreased lactate (P = .003) concentrations,

and decreased calcium:phosphorus ratio (P = .001).

Tissue concentrations of NAG detected in the

control snake are depicted (Figure 4). The liver dis-

played the highest NAG concentrations among the

tested organs, although renal tissue accounted for

the second highest concentration of NAG.

Discussion

The model used in this study was successful in

inducing renal tubular necrosis in all snakes tested.

Alternatively, a contribution of the initial renal

biopsy to the histologic lesions cannot be ruled out.

Regardless, this model enabled evaluation of the

eects of acute tubular lesions on blood parameters.

Parameters found to vary signicantly as renal

tubular pathology progressed were uric acid, ALT,

AST, total calcium, lactate, sodium, and iCa concen-

trations, as well as the calcium:phosphorus ratio.

Interpretation of these parameters remains chal-

lenging in a clinical setting as it relies on subopti-

mal reference values published in textbooks.

24,25

be diagnostically useful, blood parameters of snakes

with renal disease should have minimal overlap with

those of healthy animals. Thus, dening narrower

reference intervals for corn snakes would be very rel-

evant to minimize this overlap. At this point, no ref-

erence intervals complying with the guidelines of the

American Society of Veterinary Clinical Pathology

have been published for corn snakes. Using a single

analyzer to establish reference intervals and better

dening the seasonality and site of venipuncture

would result in narrower reference intervals. Despite

this limitation, multiple blood parameters were found

to deviate from current reference values used for

corn snakes in this model of induced tubular lesions.

Absolute hyperuricemia was detected in 4 out of

6 snakes after 11 days of daily gentamicin injections,

although this change was not statistically signicant

compared to baseline concentration. One of these

snakes had not eaten during the previous 10 days

so its hyperuricemia was not attributed to postpran-

dial changes. In the other 3 hyperuricemic snakes, a

contribution of postprandial hyperuricemia can not

Figure 2—Evolution of plasma concentration of total

calcium and calcium:phosphorus ratio in 6 corn snakes

(P guttatus) during induction of experimental tubular

necrosis using gentamicin 50 mg/kg, SC, in the caudal

third of the body. The boxes represent the quartiles,

whiskers represent the minimum and maximum, and the

line inside the box represents the median. T0 is the base-

line before starting gentamicin injection, T3 is the sec-

ond time point obtained after 3 injections of gentamicin,

and T11 is the nal time point obtained after 11 injec-

tions of gentamicin. *Signicant variations with P < .05.

Figure 3—Evolution of whole blood concentrations of

ionized calcium (iCa) and lactate measured on blood

gas panel (Heska Element POC; Epocal Inc) during

induction of experimental tubular necrosis using genta-

micin in 6 corn snakes (P guttatus). See Figure 2 for the

remainder of the key.

Figure 4—Tissue concentration of N-acetyl-beta-d-

glucosaminidase (NAG) in a 4-year-old healthy adult

female corn snake (P guttatus).

Unauthenticated | Downloaded 09/10/24 07:22 PM UTC

AJVR 7

be ruled out as uric acid can be increased for 8 days

after a meal in corn snakes

, and these snakes had

eaten 4 days prior. However, postprandial plasma

uric acid concentrations up to 2,550 μmol/L were

reported in a previous study,

while the snakes of

the present study reached uric acid concentrations

between 13,338 and 17,778 μmol/L. Thus, hyperuri-

cemia secondary to induced renal disease was sus-

pected. As expected, uric acid proved to be a poorly

sensitive marker of renal tubular lesions as hyper-

uricemia was detected in only some individuals and

at T11 only. Plasma BUN concentrations did not sig-

nicantly increase demonstrating a low sensitivity in

snakes. In addition, BUN is a very nonspecic marker

of renal disease as it increases after brumation and

in cases of dehydration in reptiles.

28,29

Similarly,

blood sodium concentration was a poorly sensitive

biomarker of renal disease. Renal tubular lesions can

be associated with decreased active sodium reab-

sorption leading to hypovolemic hyponatremia.

However, blood sodium concentration would only be

a terminal marker of renal tubular disease in snakes.

When evaluating snakes 3 days after initiating

gentamicin injections, lactate, total calcium, and iCa

concentrations and the calcium:phosphorus ratio

were found to be signicantly decreased compared to

baseline values. In addition, the calcium:phosphorus

ratio and total calcium and lactate concentrations

were signicantly correlated with the score of proxi-

mal and distal tubular lesions. Among these param-

eters, only blood iCa and lactate concentrations

were below published reference ranges

in a major-

ity of the study snakes at T3. Although 1 snake had

a lactate concentration below reference values at

the beginning of the study, this snake also showed

decreasing blood lactate concentrations over time.

As signicant decreases in blood lactate concentra-

tions were noted as early as 3 days after the begin-

ning of gentamicin injections, lactate might serve

as an early biomarker of tubular lesions. Recently,

lactate concentrations have gained interest in reptile

medicine as a prognostic indicator in rehabilitated

chelonians.

31,32

Increasing lactate concentrations

have been associated with a guarded prognosis,

while other studies

have suggested improved nest-

ing in sea turtles with hyperlactatemia. Reptiles

produce lactate during anaerobic metabolism and

are particularly prone to perform anaerobic exer-

cise compared to mammals. Chelonians are able to

sustain high lactate concentrations during hiverna-

tion,

and their renal tubular epithelium can use lac-

tate as a metabolite for glucose production. In the

present study, it is unknown if an increased uptake

of lactate could have occurred at the level of renal

tubular cells, as previously described in colubridae

or if a decreased reuptake of lactate from the pri-

mary urine could have occurred in the proximal

tubules.

Alternatively, a possible explanation for

lactate changes is that snakes’ reactivity to handling

during induction and venipuncture decreased as

renal disease progressed. Since lactate concentra-

tion is positively correlated with muscle activity in

reptiles,

decreased resistance to handling might

explain the decreased lactate concentration in later

time points. On the other hand, hyperlactatemia has

been described in box turtles with a quiet mentation,

compared to healthy conspecics.

Thus, lethargic

snakes would have been expected to display higher

lactates whereas the opposite was observed in the

present study. Alternatively, lactate may decrease

over time during anesthesia when snakes are ven-

tilated.

However, it took 6 hours of anesthesia in

rattlesnakes to reach lactate concentrations simi-

lar to those noted at T11,

while blood samples

were obtained within 10 minutes of induction in all

instances in the present study. In addition, acid-

base disturbance was restored after only 12 hours

in a previous study.

Thus, the sole eect of ven-

tilation under anesthesia to explain hypolactatemia

seems unlikely, especially in a context where blood

pH and carbon dioxide remain within normal limits.

Regardless of the cause, further studies should aim

to evaluate the prevalence of hypolactatemia in rep-

tiles with renal disease. Assessing the agreement

between various handheld lactate meters would also

be relevant, as previously performed in sea turtles.

Previous publications have listed the

calcium:phosphorus ratio as the earliest aected

parameter in cases of renal disease in snakes.

41,42

iCa

and lactate concentrations may in fact be more sen-

sitive parameters using currently available reference

intervals.

This previous statement regarding the

high sensitivity of the calcium:phosphorus ratio was

based on a retrospective study

in 12 green iguanas

published in a conference proceeding. It should be

noted that iCa concentration is not aected by egg

laying in females

and remains within similar ref-

erence intervals in Indigo snake (Drymarchon cou-

peri),

which might make it a more specic indicator

of renal disease than total calcium concentration.

However, iCa may be decreased secondary to hyper-

parathyroidism, and combined interpretation with

more specic markers of renal disease is suggested.

Surprisingly, NAG was not found to be a spe-

cic biomarker for corn snake renal tissue as it was

detected in high concentrations in the liver and intes-

tine in this species. In pigeons, renal NAG concentra-

tion has been shown to be at least 6 times higher

than in other tissues, including liver, intestine, and

pancreas.

This dierence highlights the importance

of species-specic studies to evaluate enzymatic tis-

sular distribution.

In the present study, SDMA proved to be an

insensitive marker of tubular necrosis in corn snakes.

In contrast, a similar experimental model using

gentamicin to induce acute tubular necrosis in rats

resulted in an increased serum SDMA concentra-

tion.

A previous retrospective study hypothesized

that SDMA could be an early indicator of renal dis-

ease in chelonians

; this conclusion was based on

the results obtained in 28 individuals showing a

correlation between elevated uric acid and SDMA

concentrations. However, no renal histology was

available, and it is, therefore, possible that this

conclusion was erroneous. Further studies are war-

ranted to determine if SDMA concentrations could

Unauthenticated | Downloaded 09/10/24 07:22 PM UTC

8 AJVR

increase in reptiles aected by glomerular disease

instead of renal tubular pathology or in reptiles with

chronic versus acute renal disease or whether its

metabolism diers between chelonians and snakes.

Pending more studies, SDMA concentration should

not be used as a marker of renal disease in reptiles.

Other diagnostic options are recommended such as

renal biopsy.

42,47

Despite hepatic steatosis being noted in all

snakes, AST and ALT did not increase signicantly,

which was surprising as these enzymes have been

found in high quantities in colubrid snake renal and

hepatic tissue.

No signicant increase in plasma

GGT concentrations was detected at any time point

in the present study. This enzyme is thought to be

highly specic as it is detected only in the lungs and

kidneys of snakes and box turtles.

13,48

However, the

presence of an enzyme in renal tissue does not neces-

sarily correlate with its elevation in the bloodstream

after tissue damage, as previously demonstrated for

glutamate dehydrogenase in birds.

Similarly, GGT

may be excreted in the urine rather than increasing

in the bloodstream. Although no data are available

in reptiles, GGT has been detected in bird urine.

Further studies should evaluate GGT concentration

in urine in healthy versus renal insucient reptiles.

Limitations of the present study include the

small number of individuals included, but ethical

considerations precluded enrollment of a large snake

cohort. In particular, more healthy individuals could

have been used to evaluate the NAG tissue distri-

bution and evaluate the interindividual variability.

The variations of parameters observed in this study

could be related to renal lesions or could potentially

be associated with repeated handling or other fac-

tors in the absence of a control group. In addition,

signicant disagreement has been described for

several analytes measured by blood gas analyzers

compared to gold-standard techniques in reptiles.

Further studies should aim to evaluate the analyti-

cal performance of blood gas analyzers in reptiles. In

dogs and cats, other novel markers of renal disease

include clusterin, cystatin B, and inosine.

8,52

Serum

inosine concentration has been shown to decrease in

a model of gentamicin-induced renal disease in dogs

as it is a marker of tubular epithelial cells.

However,

this marker was not available at the time of this study.

In conclusion, iCa and lactate concentrations were

the most sensitive early indicators of renal tubular

pathology in corn snakes among tested parameters.

Although still within reference values, total calcium

concentrations and the calcium:phosphorus ratio

decreased signicantly 3 days after induction of

renal disease compared to baseline values. Further

studies are needed to establish reference intervals

for iCa and lactate in colubrid snakes.

Acknowledgments

We thank Tristan Juette, statistician for his advice,

Amélie Aduriz for her assistance, and Corinne Généreux from

the chromatography laboratory.

Disclosures

The authors have nothing to disclose. No AI-assisted

technologies were used in the generation of this manuscript.

Funding

This project was funded by a grant from the Natural

Sciences and Engineering Research Council of Canada.

Funding was also provided by the CHUV Fund, Faculté de

Médecine Vétérinaire, Université de Montréal. We thank Hagen

for the donation of the terraria and lights used in this project.

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Supplementary Materials

Supplementary materials are posted online at the jour-

nal website: avmajournals.avma.org

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