Differences between renal effects of venom from two Bothrops jararaca populations from southeastern and southern Brazil

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Short communication

Differences between renal effects of venom from two

Bothrops

jararaca

populations from southeastern and southern Brazil

Roberta Jeane Bezerra Jorge

a,*

, Ant

onio Rafael Coelho Jorge

^

b

,

Ramon R

oseo Paula Pessoa Bezerra de Menezes

b

, Clarissa Perdig

~

ao Mello

c

,

Danya Bandeira Lima

c

, Jo

~

ao Alison de Moraes Silveira

b

, Natacha Teresa Queiroz Alves

b

,

Aline Diogo Marinho

b

, Rafael Matos Ximenes

d

, Carlos Corr

^

ea-Netto

e

,

Larissa Gonçalves Machado

e

, Russolina Benedeta Zingali

e

, Alice Maria Costa Martins

c

,

Helena Serra Azul Monteiro

b

a_{Centro Acad}_^_{emico de Vit}_{oria, Universidade Federal de Pernambuco, Vit}_{oria de Santo Ant}_~_{ao, Pernambuco, Brazil} b_{Departamento de Fisiologia e Farmacologia, Universidade Federal do Cear}_{a, Fortaleza, Ceara, Brazil}

c_{Departmento de An}_{alises Clínicas e Toxicol} _{ogicas, Universidade Federal do Cear}_{a, Fortaleza, Ceara, Brazil} d_{Departmento de Antibi}_{oticos, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil} e_{Instituto Nacional de Biologia Estrutural e Bioimagem, Instituto de Bioquímica M}_{edica Leopoldo de Meis}

eUniversidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

a r t i c l e

i n f o

Article history: Received 17 August 2016 Received in revised form 11 November 2016 Accepted 16 November 2016 Available online 17 November 2016

Keywords:

Bothrops jararacavenom Renal effects

MDCK

Isolated kidney perfusion

a b s t r a c t

Components from animal venoms may vary according to the snake's age, gender and region of origin. Recently, we performed a proteomic analysis ofBothrops jararacavenom from southern (BjSv) and southeastern (BjSEv) Brazil, showing differences in the venom composition, as well as its biological ac-tivity. To continue the study, we report in this short communication the different effects induced by the BjSEvandBjSvon isolated kidney and MDCK renal cells.BjSEvdecreased perfusion pressure (PP) and renal vascular resistance (RVR) and increased urinaryflow (UF) and glomerularfiltration rate (GFR), whileBjSvdid not alter PP and RVR and reduced UF and GFR. Both types of venom, more expressively BjSEv, reduced %TNaþ_{, %TK}þ_{and %Cl . In MDCK cells, the two types of venom showed cytotoxicity with} IC50of 1.22mg/mL forBjSvand 1.18mg/mL forBjSEvand caused different profiles of cell death, withBjSv being more necrotic. In conclusion, we suggest thatBjSvis more nephrotoxic thanBjSEv.

1. Introduction

Snake venoms are a complex mixture of proteins, peptides and small inorganic molecules. Clinical manifestations of bothropic accidents consist in various local and systemic effects, mainly characterized by severe coagulopathy, necrosis, edema, sponta-neous bleeding and, in some cases, compartment syndrome (França et al., 2003; Santoro et al., 2008). Systemic complications occur in the absence of antivenom administration, leading to more severe cases associated with intense bleeding, shock and acute kidney injury (AKI), which are closely related to death (Ribeiro and Jorge, 1997; De Castro et al., 2004; Sgrignolli et al., 2011).

There are many cases of snake envenomation annually in Brazil,

most of them associated with theBothropsgenus (Queiroz et al.,

2008). Bothrops jararaca (Wied-Neuwied, 1824) is a medically-relevant, semi-arboreal pit viper, found from the state of Bahia to Rio Grande do Sul in Brazil and in parts of Paraguay and Argentina. This wide geographical distribution range is associated with the most populated areas in Brazil (Sazima, 1992).

Recently, a combined proteomic study was conducted using

venom pools from the outermost points ofB. jararacageographical

distribution in Brazil. These representative venom pools of the

southeastern and southern populations of B. jararaca revealed

distinct quantitative and some qualitative overall toxin composi-tions of proteomes and transcriptomes. The different composicomposi-tions

of the venoms ofB. jararacafrom the south and southeast have

demonstrated different biological actions, such as coagulant,

pro-teolytic and of phospholipase A2actions (Gonçalves-Machado et al.,

*Corresponding author.

E-mail address:robertajeanebj@gmail.com(R.J.B. Jorge).

Contents lists available atScienceDirect

Toxicon

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t o x i c o n

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2016). To continue this study, we report in this short communica-tion the differences in renal effects induced by both venoms on an isolated kidney perfusion model and cytotoxicity on MDCK renal epithelial cells.

2. Material and methods

B. jararacavenom samples from the southeastern snake

popu-lation were collected from a number (n¼_{22) of adult specimens}

captured in the following states of Brazil: Rio de Janeiro, Espirito Santo and Minas Gerais. The specimens were maintained at the

serpentarium of Vital Brazil Institute (Niteroi, Rio de Janeiro, Brazil)

under IBAMA registration n. 26354-9, while the venom samples of

adult specimens of B. jararaca(n¼22) from the southern

pop-ulations were captured in localities of the state of Rio Grande do Sul (RS) and kept in captivity in the serpentarium of Center of Ophi-ology of Porto Alegre, Rio Grande do Sul, Brazil, under IBAMA registration n. 1/43/1999/000764-9.

We performed kidney perfusion tests to evaluate the renal effects with no interference of systemic factors. In this assay, adult male Wistar rats (260e320 g) were fasted for 24 h with free access to water. The rats were anesthetized with sodium pentobarbitone (50 mg/kg, i.p) followed by careful dissection of the right kidney. The right renal artery was cannulated via the mesenteric artery without

interrupting blood ﬂow, as described by Bowman (1970). The

perfusate consisted of modiﬁed Krebs-Henseleit solution (MKHS)

consisting of (in mmol/L): 114.00 NaCl, 4.96 KCl, 1.24 KH2PO4, 0.5

MgSO4.7H2O, 2.10 CaCl2and 24.99 NaHCO3. Bovine serum albumin

(BSA 6 g%; fraction V), urea (0.075 g), inulin (0.075 g) and glucose

(0.15 g) were added to the solution, resulting in aﬁnal perfusate

volume of 100 mL. The pH was adjusted to 7.4. In each experiment, 100 mL of MKHS were recirculated for 120 min. The perfusion pressure (PP) was measured at the tip of the stainless steel cannula in the renal artery. Urine and perfusate samples were collected at 10 min-intervals for analysis of sodium, potassium and chloride levels by ion-selective electrodes (RapidChem 744, Bayer Diagnostic, UK);

inulin, as described byWalser et al. (1955)and modiﬁed byFonteles

et al. (1983); and osmolality, which was measured in a vapor

pres-sure osmometer (Wescor 5100C, USA).BjSvorBjSEv(10

mg/mL) was

added to the system 30 min after the beginning of each perfusion. The perfusion pressure (PP), renal vascular resistance (RVR), urinary

ﬂow (UF), glomerularﬁltration rate (GFR), the percentage of sodium

(%TNaþ), potassium (%TKþ) and chloride (%TCl-) tubular transport

were determined (Martinez-Maldonado and Opava-Stitzer, 1978). The results were compared to the internal control group, at early

30 min in each experiment (n¼6). In this experiment, both right and

left kidneys wereﬁxed with formaldehyde until subsequent

histo-logical analysis. The fragments were subjected to dehydration,

diaphanization and then cut into 3

mm thick slices. Then,

hematoxylin-eosin (HE) staining was performed and the slides analyzed using an optical microscope.

We also performed assays in cells to evaluate renal cytotoxicity. Epithelial MadineDarby Canine Kidney (MDCK) cells were cultured in RPMI 1640 medium (RPMI, Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St.

Louis, MO, USA), 1% penicillin/streptomycin solution at 37_{C, 5%}

CO2. MDCK cells were cultured and treated with different

concen-trations ofBjSvorBjSEv(3.12; 1.56; 0.78; 0.39

mg/mL), incubated at

37_{C for 24 h and mitochondrial function was measured by MTT}

colorimetric assay. MTT reagent (Sigma-Aldrich, St. Louis, MO, USA;

5 mg/mL in PBS) was added and incubated for 4 h at 37_{C. Finally,}

the medium was removed and the precipitated formazan crystals were dissolved in 10% Sodium dodecyl sulfate (Vetec, Rio de Janeiro, RJ, Brazil). After 17 h, absorbance was read at 570 nm in a

micro-plate reader (Biochrom®

Asys Expert Plus). Cell viability was

calculated in comparison with the control group. The IC50 was

determined by non-linear regression (Mosmann, 1983).

Finally, the cell death proﬁle was evaluated with Annexin V-FITC

(AX) and 7AAD staining. Cells treated with IC50and 2IC₅₀ofBjSv

(1.22 and 2.44

mg/mL) and

BjSEv(1.18 and 2.36

mg/mL) incubated

during 12 h were stained with ﬂuorescein isothiocyanate

(FITC)-conjugated to annexin V/7AAD according to the manufacturer's instructions (BD Pharmingen, CA, USA). The population of

AX-7AAD-viable cells was evaluated by ﬂow cytometry. Data were

collected in a FACSCaliburﬂow cytometer and analyzed using Cell

Quest software (Becton-Dickinson, Mountain View, CA, USA).

Statistical analysis shows values as mean± _{S.E.M of triplicate}

values for each cell experiment and hexaplicate values for perfused

renal experiments. The signiﬁcance of the difference between the

experimental and control groups was assessed by one-way analysis

of variance (ANOVA). Signiﬁcance was deﬁned as p<0.05.

3. Results and discussion

Bothrops jararacavenom consists of a mixture of molecules, such as metalloproteinases, disintegrin, vasoactive peptides (VAP; including BPPs, bradykinin-potentiating peptides), phospholipase

A2(PLA2), serine proteinases (Serprot) and L-amino-acid oxidases

(LAAO). The relative occurrence (in percentage of total venom

proteins) of toxins from venom pools of B. jararaca populations

from the Southeastern and Southern Atlantic forest regions disclose

many differences.BjSEvhas 35% of metalloproteinases, 7% of

dis-integrin, 16% of VAP, 4% of PLA2, 14% of Serprot; whileBjSvhas 10%

of metalloproteinases, 0.2% of disintegrin, 22% of VAP, 20.2% of PLA2,

28% of Serprot, and (Gonçalves-Machado et al., 2016).

The intrinsic toxicity of toxins and their abundance in venom are relevant when evaluating their potential medical impact (Laustsen et al., 2015). Phospholipases and metalloproteinases, among several enzymes, are believed to contribute to renal toxicity (Sitprija and Sitprija, 2012). TheBjSEv has more metalloproteinases, while the

BjSv has a higher amount of venom PLA2(Gonçalves-Machado et al.,

2016).

The effects of Bothrops venoms on isolated kidney have been

previously demonstrated. The Bothrops leucurus (Morais et al.,

2013),Bothrops marajoensis(Evangelista et al., 2010) and Bothro-poides pauloensis(Marinho et al., 2015) cause reduction in PP, RVR, UF, GFR and tubular transport of electrolytes. Furthermore, Bothrops moojeni(Barbosa et al., 2002),Bothropoides erythromelas

(Martins et al., 2005) andBothrops jararacussuvenoms (Havt et al.,

2001) decrease PP, RVR, tubular transport of electrolytes and in-crease UF and GFR.

Our results showed different proﬁles of renal alterations on

isolated kidney.BjSEvdecreased PP (Fig. 1A) and RVR (Fig. 1B) at

60 min, increased UF (Fig. 1C) at 90 and 120 min and GFR (Fig. 1D) at

120 min. BjSv, in turn, did not alter PP, RVR (Fig. 1A and B) and

reduced UF and GFR (Fig. 1C and D) at 60, 90 and 120 min. Both venoms caused renal tubular dysfunction by altering the

percent-age of tubular transport of electrolytes. They reduced %TNaþ

(Fig. 1E), %TKþ

(Fig. 1F) and %TCl (Fig. 1G) at 60, 90 and 120 min,

with the reduction of %TNaþ

and %TCl induced byBjSEvbeing

more signiﬁcant.

Bothrops erythromelas venom has shown alterations in the perfused kidney (Martins et al., 2005), similar to alterations

observed inBjSEv.Metalloproteinases are the major component in

B. erythromelasvenom (Jorge et al., 2015) andBjSEv,suggesting this

fraction may be responsible for part of the renal alterations.BjSEv

showed a diuretic effect, in turn,BjSvshowed a sharp decline of

glomerularﬁltration rate together with tubular dysfunction.

Renal pathological changes in patients with AKI caused by

snakebites include tubular necrosis, cortical necrosis,

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glomerulonephritis and vasculitis (Kanjanabuch and Sitprija, 2008; Sitprija and Sitprija, 2012). Histopathological analyses of kidneys

from rats exposed toB. jararacavenom also showed tubular necrosis

(Monteiro and Fonteles, 1999). In this work, the histological analysis

Fig. 1.Effects of theBothrops jararacavenom from southeastern (BjSEv) and southern Brazil (BjSv) (10mg/mL) on perfusion pressure (PP;A); renal vascular resistance (RVR;B); urinaryﬂow (UF;C); glomerularﬁltration rate (GFR;D); percentage of sodium tubular transport (%TNaþ

;E); percentage of potassium tubular transport (%TKþ

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of the kidneys perfused withBjSvdemonstrated higher toxicity. The

kidneys treated withBjSvshowed abundant proteinaceous material

in the urinary space and tubules, tubular dilation and vacuolar

hydropic degeneration.BjSEvalso showed deposition of

proteina-ceous material in renal tubules, tubular dilation and vacuolar hydropic degeneration, but the glomeruli showed no deposition in the urinary space (Fig. 2).

The loss of tubular epithelial cell function could lead to tubular

dysfunction (Dantas et al., 2015). Both BjSv and BjSEv showed

cytotoxicity on MDCK distal tubular epithelial renal cells. MDCK cells constitute a very-well established cell line and have been extensively employed in the investigation of several cell processes, including epithelial transport and cell response to toxic agents and venoms (Peixoto and Collares-Buzato, 2005; Kusma et al., 2008).

The concentration capable to kill 50% of MDCK cell of BjSvwas

1.22

mg/mL (Fig. 3A) and 1.18

mg/mL for

BjSEv(Fig. 3D). Despite the

fact that IC50were very similar,BjSv(Fig. 3B and C) showed a higher

necrotic proﬁle thanBjSEvinﬂow cytometry (3E and 3F).

Cellular injury can result from enzyme action, especially that of

PLA2and proteases on cell membrane. Cell injury caused by PLA2

consists of hemolysis, rhabdomyolysis, neuronal damage, renal tubular damage and endothelial necrosis, while metalloproteinases

can cause destruction of the cytoskeleton, leading to cell damage, cell adhesion disruption and apoptosis (Sitprija and Sitprija, 2012). Necrosis is characterized by rapid cytoplasmic swelling, culmi-nating in plasma membrane rupture and organelle breakdown

(Vanden Berghe et al., 2013).BjSvshowed an increase of 7AADþ/

AX- and 7AADþ_{/AX-, a predominantly necrotic pro}_ﬁ_{le, which can}

be related to high concentrations of PLA2in its composition. PLA2is

toxic due to its effects on biological membranes through charge and van der Waals interaction, leading to membrane destabilization (Sitprija and Sitprija, 2012).

Apoptosis is a programmed cell death pathway, signaled by phosphatidylserine externalization. Apoptosis could become sec-ondary necrosis in the absence of phagocytosis (Vanden Berghe et al., 2013).BjSEvshowed only increase of 7AADþ/AXþ, a proﬁle

of secondary necrosis, which can be related to the high concen-trations of metalloproteinases in its composition.

Bothrops venom such as,Bothrops pauloensis,Bothrops diporus

andBothrops pirajaivenoms have shown cytotoxic activity in tumor cells and the cytotoxic mechanism related to both necrosis and

apoptosis (Jorge et al., 2011).Bothrops leucurus(Morais et al., 2013),

B. erythromelas(De Sousa et al., 2016) andB. pauloensis(Marinho et al., 2015) venoms have shown an apoptotic proﬁle, while

Fig. 2.Histological analysis of the kidneys perfused withBjSvandBjSEv. No changes were observed in glomeruli and tubules in controls: kidneys perfused with Krebs-Henseleit solution (A) and kidneys not perfused (B). Kidneys perfused withBjSEv(10mg/mL) displayed intratubular protein deposits (1) and vacuolar hydropic tubular degeneration (2) (C and D). Kidneys perfused withBjSv(10mg/mL) showed protein deposition in the urinary space (3), intratubular protein deposits (4) and vacuolar hydropic tubular degeneration (5) (E and F). 400 X. H/E.

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Bothrops insularis(Mello et al., 2014) venom has shown a necrotic

proﬁle predominantly on MDCK cells (Grazziotin et al., 2006).

Similar concentrations ofBjSEvandBjSvvenoms can result in

different proﬁles of cell death, in which the one induced byBjSvis

quicker and could be related to higher toxicity observed at 120 min of renal perfusion. It indicates the necessity of quickly establishing

the treatment of patients bitten byB jararacafrom south snakes.

The difference in nephrotoxicity found in these two bothrops venoms could be related to quantitative and qualitative differences

in their components.BjSvis rich in PLA2(Lys49-PLA2), which is an

exclusive component of this venom (Gonçalves-Machado et al., 2016). PLA2 plays an important role in cell injury through the

ability of mediating inﬂammatory response, catalyzing the

hydro-lysis of phosphoglycerides and leading to membrane damage (Sitprija and Sitprija, 2012). The presence of the strategic residues

known to constitute the Ca2þ_{-binding loop of PLA}

2, such as Lys-49,

could be directly or indirectly involved in its catalytic activity

(Pereira et al., 1998). In the present study, BjSv showed more

toxicity in isolated kidney and quicker cell death pathway in MDCK

cells thanBjSEv. This fact could be related to high concentrations of

Lys49-PLA2inBjSv.

In conclusion, both types of venoms of the same species from different geographical regions showed differences regarding effects

on renal function parameters and cytotoxicity, being BjSv more

nephrotoxicity thanBjSEv. However, further studies with the

iso-lated toxins are needed to elucidate which toxin is involved in the pathophysiology of renal injury.

Ethical statement

This study was approved by the Ethics Committee on Animal Research (CEUA/UFPE) of Universidade Federal de Pernambuco (Authorization Number: 129/15).

Acknowledgements

We thank Moema Leit~ao de Araújo and Maria Lúcia Machado

Alves from Regional Center of Ophiology of Porto Alegre (NOPA)

and Leonora Brazil Mas from Vital Brazil Institute (IVB) for donating

the venoms. We thank Maria Silvia Helena Freire de França

(Uni-versidade Federal do Ceara) for their excellent technical assistance.

We thank National Council of Technological and Scientiﬁc

Devel-opment (CNPq) and Coordination for Enhancement of Higher

Ed-ucation Personnel (CAPES) for theirﬁnancial support.

Transparency document

Transparency document related to this article can be found

online athttp://dx.doi.org/10.1016/j.toxicon.2016.11.249.

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