Toxicon 165 (2019) 40–46

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Toxicon

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Nephrotoxicity induced by the venom of hypnale from : Studies on isolated perfused rat kidney and renal tubular cell lines T

Mauren Villaltaa, Tiago Lima Sampaiob, Ramon Róseo Paula Pessoa Bezerra de Menezesb, Dânya Bandeira Limab, Antônio Rafael Coelho Jorgec, Renata Sousa Alvesb, Helena Serra Azul Monteiroc, Indika Gawarammanad, Kalana Maduwaged, Roy Malleappahe, ∗ Guillermo Leóna, Alice M.C. Martinsb, José María Gutiérreza, a Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica b Department of Clinical and Toxicological Analysis, Federal University of Ceará, Fortaleza, Ceará, Brazil c Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceara, Brazil d Faculty of Medicine, University of Peradeniya, Sri Lanka e Venom Research International (AVRI), California, USA

ARTICLE INFO ABSTRACT

Keywords: The hump-nosed Hypnale hypnale is responsible for a high number of snakebite cases in southwestern Hypnale hypnale and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a Hump-nosed pit viper growing body of evidence indicating that these envenomings may be associated with systemic alterations, in- Nephrotoxicity cluding acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused Cytotoxicity isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The Renal tubular cells venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these ex- perimental models, in agreement with clinical observations.

1. Introduction mild coagulopathy, bleeding, thrombotic microangiopathy, acute kidney injury (AKI), and chronic kidney injury (CKI) (de Silva et al., Snakebite envenoming is a serious medical hazard in the Indian 1994; Kularatne and Ratnatunga, 1999; Joseph et al., 2007; Ariaratnam peninsula and Sri Lanka (Warrell, 1995). In addition to the traditional et al., 2008, 2009; Kularatne et al., 2011; Herath et al., 2012; so-called ‘big four’ species (Daboia russelii, Echis carinatus, Naja naja and Maduwage et al., 2013; Karunarathne et al., 2013; Shivanthan et al., Bungarus caeruleus), which are responsible for the majority and most 2014; Rathnayaka et al., 2018, 2019a, 2019b). AKI is one of the main severe snakebite envenomings, the hump-nosed viper, Hypnale hypnale, causes of morbidity and mortality in envenomings inflicted by many causes a high number of snakebites in this region (Warrell, 1995; species (Albuquerque et al., 2014). Ariaratnam et al., 2008, 2009; Shivanthan et al., 2014). In the past, H. The pathogenesis of AKI induced by the venom of H. hypnale has hypnale was considered as incapable of inflicting severe envenomings in remained elusive, although the effects of this venom in the kidney at humans, but growing clinical evidence clearly shows that this snake is experimental level include damage to proximal convoluted tubules able to cause systemic manifestations. (Gunatilake et al., 2003; Silva et al., 2012; Tan et al., 2012). As with The majority of envenomings by H. hypnale are characterized by other snake venoms that affect the kidney, it is likely that ne- only local effects, i.e. edema of variable extension and pain (Warrell, phrotoxicity by H. hypnale is of multi-factorial nature, probably invol- 1995; Ariaratnam et al., 2008; Kularatne et al., 2011). In addition, a ving mechanisms such as (a) renal ischemia secondary to hemodynamic number of cases also develop systemic effects of envenoming, such as disturbances, (b) thrombotic microangiopathy and hemolytic

∗ Corresponding author. E-mail address: [email protected] (J.M. Gutiérrez). https://doi.org/10.1016/j.toxicon.2019.04.014 Received 1 March 2019; Received in revised form 19 April 2019; Accepted 22 April 2019 Available online 26 April 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved. M. Villalta, et al. Toxicon 165 (2019) 40–46 microangiopathic anemia, (c) tubular cell toxicity induced by he- 2.3. Cytotoxicity of venom on kidney-derived cells moglobin accumulated in the tubular lumen, and (d) direct nephrotoxic action of venom components (Sitprija and Sitprija, 2012). In order to Cytotoxicity of venom was tested in cell culture using the cell lines study the nephrotoxic action of H. hypnale venom at the experimental LLC-MK2 (Monkey Kidney epithelial cell line) and MDCK (Madin-Darby level, the present study was carried out by assessing the effect of this Canine Kidney epithelial cells), as previously described (Morais et al., venom on a perfused isolated rat kidney system and on two different 2013; Mello et al., 2014). These cells correspond to proximal and distal renal tubular cell lines in culture. renal tubular cells, respectively. Cells were grown in DMEM supple-

mented with 10% bovine fetal serum, under 5% CO2 at 37 °C. Upon confluence, cells were grown in DMEM without fetal serum for 24 h. 2. Materials and methods Then, the confluent monolayers were treated with 10% trypsin and cells were grown in 96 well plates at a seeding density of 105 cells/well. Cells 2.1. Venom were left to adhere to the plate overnight and were then treated with venom. For this, solutions of various venom concentrations were added A pool of venom from 35 adult specimens of H. hypnale was used in to wells and incubated for 24 h. Control wells were treated with 0.12 M this study. Venom was obtained from specimens collected in the field in NaCl, 0.04 M phosphate, pH 7.2 (PBS) solution. Assays were run in four provinces of Sri Lanka [Central Province (wet zone); North triplicates. Cytotoxicity was assessed by reduction of 3-(4,5-di- Western province (dry and intermediate zones); Southern province (wet methyltiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma, St. zone); Sabaragamuaga province (wet zone)] following the granting of a Louis, MO, USA). Ten microliters of MTT solution (2.5 mg/mL in PBS) collecting permit from the Department of Wildlife Conservations, Sri were added to each well, at the time intervals indicated, and incubated Lanka (WL/3/2/7), and kept at the serpentarium of Animal Venom at 37 °C for 4 h. The medium was removed and the precipitated for- Research International (AVRI) in Sri Lanka. The male: female ratio was mazan crystals were dissolved in 10% sodium dodecyl sulphate (SDS) in 20 : 15. Upon collection, venom was frozen and then freeze-dried, and 0.01 N HCl. After 17 h of incubation, absorbances at 570 nm were re- ® stored at −20 °C until used. The identification of the specimens as corded in a microplate reader (Biochrom Asys Expert Plus). Cell via- belonging to H. hypnale was based on the keys previously published by bility was estimated in comparison with the control group. The Median

Maduwage et al. (2009). Cytotoxic Concentration (CC50), i.e. the venom concentration that in- duced 50% cytotoxicity, was estimated using the program GraphPad Prism. 2.2. Kidney perfusion experiments 2.4. Annexin V-FITC staining Adult male Wistar rats (260–320 g) were fasted for 24 h with free water access. They were anesthetized with sodium pento-barbitone Staining of cells with Annexin was used to assess apoptosis. LLC- (50 mg/kg, i.p.) and, after dissection of the right kidney, the renal ar- MK2 or MDCK cells were seeded at 105 cells/well, as described, and tery was cannulated through the mesenteric artery. The kidney was incubated for 24 h at 37 °C. Then, solutions of two venom concentra- perfused with a modified Krebs-Henseleit solution (MKHS) with the tions, corresponding to 1 and 2 CC50, were added. Controls included following composition (in mmol/L): 114 NaCl, 4.96 KCl, 1.24 KH2PO4, cells incubated with PBS. After an incubation period of 24 h, cells were 0.5 MgSO4.7 H2O, 2.10 CaCl2, 25 NaHCO3. Bovine serum albumin trypsinized, centrifuged at 500 g for 5 min, and the supernatant was (BSA, 6% w/v), urea (0.75 mg/mL), and glucose (1.5 mg/mL) were discarded. This step was repeated twice using PBS. The cellular pellet added to the solution, and the pH was adjusted to 7.4. Thereafter, inulin was labeled with the Annexin/7AAD using the kit BD Pharmingen PE (0.75 mg/mL) was added. In each experiment, 100 mL of MKHS were Annexin Apoptosis Kit I (BD Pharmigen, CA, USA). Controls included recirculated during 120 min through the system. The perfusion pressure cells incubated with either Annexin/7AAD alone. Samples were in- was kept between 120 and 140 mmHg. Initially, perfusion was per- cubated for 30 min at room temperature in the dark. Then, samples formed without venom for 30 min in order to have basal levels of each were analyzed by flow cytometry in a FACS Calibur (Becton-Dickinson, determination. After this, H. hypnale venom (10 μg/mL) was added to Mountain View, CA, USA) and analyzed using a Cell Quest software the system and changes were observed. Samples of urine and perfusate (Becton-Dickinson). were collected every 10 min and perfusion pressure (mmHg) and flow (L/min) were recorded every 5 min. A control group perfused with 2.5. Rhodamine staining MKHS only was followed during 120 min. Five rats were used for each treatment. Staining of cells with Rhodamine 123 (Sigma) was used to assess the Perfusion pressure (PP) was determined at the tip of the stainless mitochondrial membrane potential (Baracca et al., 2003). Cells were steel cannula in the renal artery. Osmolality was measured by using a incubated with venom solutions, as described above. Then, after cen- PZL-1000 osmometer (Wescor 5100C, USA), and concentrations of so- trifugation, the cellular pellet were resuspended in 100 μL PBS, and dium, potassium and chloride were determined by using a 9180 100 μL of a Rhodamine 123, dissolved in PBS, were added. After an Electrolyte Analizer (Rapidchem 744, Bayer Diagnostic, UK). Inulin incubation of 30 min at room temperature in the dark, 1 mL of PBS was concentration was quantified according to Walser et al. (1955) as added and the preparation was centrifuged at 500 g for 5 min. The modified by Fonteles et al. (1983). The following renal functional pellets were resuspended in 500 μL PBS and flow cytometry analysis parameters were determined: perfusion pressure (PP), renal vascular was then carried out. resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and the percentages of sodium (%TNa+), potassium (%TK+), and 2.6. DCFH-DA staining − chloride (%TCl ) total tubular transport and proximal tubular trans- port (Martínez-Maldonado and Opava-Stitzer, 1978). Results were Staining of cells with 2,7-dichlorodihydro-fluorescein diacetate compared to the internal control group corresponding to the values at (DCFH-DA) (Sigma) was used to measure the cellular levels of reactive the 30 min perfusion period prior to venom introduction into the oxygen species, as described by Morais et al. (2015). Cells were in- system. The protocol was approved the Ethics Committee on Animal cubated with venom, as described above. After incubation, cells were Research (CEPA) of the Federal University of Ceara (Permit Number trypsinized and centrifuged at 500 g for 5 min. After several washing 79/08). steps, cells were resuspended in PBS containing 5 μM DCFH-DA, and incubated for 30 min. Controls included cell suspensions in which

41 M. Villalta, et al. Toxicon 165 (2019) 40–46

DCFH-DA was not added. After incubation, cells were analyzed by flow 4. Discussion cytometry. The data based on the FL1 channel were analyzed with the Cell Quest program. The majority of envenomings caused by H. hypnale are characterized only by local effects, i.e. pain and edema, but a growing body of pub- lished clinical evidence demonstrates that this viperid species is also 2.7. Statistical analyses capable of inducing severe envenomings characterized by systemic manifestations, such as mild coagulopathy, hemorrhage, thrombotic Results were expressed as mean ± S.E.M. of three or five replicates, microangiopathy, and AKI (de Silva et al., 1994; Ariaratnam et al., in the case of cell culture and renal perfusion experiments, respectively. 2008, 2009; Kularatne et al., 2011; Herath et al., 2012; Maduwage The significance of the differences between mean values was assessed et al., 2013; Shivanthan et al., 2014; Rathnayaka et al., 2018, 2019a, by ANOVA, using a Dunnett post-hoc test to compare pairs of means, ® 2019b). The pathogenesis of AKI in envenomings by this and other using the program GraphPad Prism 5.0. Values of p < 0.05 were snake species is likely to be multifactorial (Sitprija and Sitprija, 2012). considered statistically significant. The clinical expression of AKI is an oliguric or anuric state associated with acute tubular necrosis after envenoming (Albuquerque et al., 3. Results 2014). Previous studies have assessed the effects of venoms of various 3.1. Effects of venom on kidney functional parameters species of of the Bothrops in the isolated kidney model used in the present investigation (Evangelista et al., 2010; Morais et al., Control experiments carried out on kidneys perfused with Krebs- 2013; Marinho et al., 2015). Bothrops sp venoms induced a drop in Henseleit solution only did not show any significant changes in the perfusion pressure, renal vascular resistance, urinary flow, glomerular parameters evaluated along the 120 min observation time. The perfu- filtration rate, and sodium tubular transport. Bothrops moojeni and B. sion of a venom solution (10 μg/mL) in the isolated kidney preparation jararacussu venoms showed similar effects, but caused an increase in induced changes, as compared to controls, in almost all functional urinary flow (Havt et al., 2001; Barbosa et al., 2002). In the present parameters evaluated. There was a significant reduction in perfusion work, H. hypnale venom promoted a reduction of perfusion pressure, pressure at 90 and 120 min (PP; Fig. 1A), renal vascular resistance at 90 renal vascular resistance, sodium and chloride tubular transport, and an and 120 min (RVR; Fig. 1B), percentage of transported sodium at 90 initial drop of glomerular filtration rate and urinary flow, followed by and 120 min (%Na+; Fig. 1E), percentage of proximal tubule trans- an increment in these two parameters. Our findings underscore the ported sodium at 60, 90 and 120 min (%pNa+; Fig. 1F), percentage of ability of H. hypnale venom to induce renal damage. Although the − transported chloride at 120 min (%Cl ; Fig. 1I), and percentage of mechanisms behind these alterations remain to be elucidated, our study − proximal tubule transported chloride at 120 min (%pCl ; Fig. 1J). provides experimental evidence that this venom alters functional Regarding urinary flow, there was a slight initial reduction at 60 and parameters, such as reduction on RVR and PP. These alterations could 90 min (UF; Fig. 1C), followed by an increment at 120 min, whereas the lead to renal ischemia secondary to hemodynamic disturbances. The glomerular filtration rate had a reduction at 60 and 90 min (GFR; pathogenesis of AKI in envenomings by H. hypnale may be also medi- Fig. 1D), followed by recovery at 120 min. Of the evaluated parameters, ated through the formation of microthrombi, i.e. thrombotic micro- only the percentage of transported potassium was not altered upon angiopahy, which contribute to renal ischemia (Rathnayaka et al., infusion of the venoms (Fig. 1G and H). 2019b). The infusion of H. hypnale venom in the isolated rat kidney caused alterations in practically all the parameters investigated, hence under- 3.2. Cytotoxicity on kidney-derived cells scoring a widespread effect of the venom on renal function. Moreover, our observations suggest that this venom affects the function of both the When cytotoxicity of venom on two kidney-derived cell lines was tubular cells and the glomeruli, as evidenced by electrolyte transport assessed by the MTT assay, a dose- and time-dependent cytotoxic effect and glomerular filtration rate. Previously, H. hypnale venom had been was observed in both cell lines. The dose-dependency was more evident shown to induce AKI in experimental models, as revealed by increments when incubation was carried out for 24 h and, therefore, the estimation in serum urea and creatinine concentrations, hematuria, proteinuria, of the Median Cytotoxic Concentration (CC ) was made at this time 50 and by histolopathogical changes in the kidney (Gunatilake et al., 2003; interval. CC s corresponded to 9.0 ± 2.9 μg/mL and 19.7 ± 12 μg/ 50 Silva et al., 2012; Tan et al., 2012). Our observations on the effect of mL μg/mL for LLC-MK2 and MDCK cell lines, respectively. Thus, venom this venom on sodium tubular transport is in agreement with a recent was cytotoxic for cell lines derived from proximal and distal renal clinical observation of a patient who developed urinary sodium loss tubular cells. associated with hyponatremia (de Silva et al., 2018). H. hypnale venom was cytotoxic to both renal tubular cell types 3.3. Evaluation of cell death mechanisms tested in culture (MDCK and LLC-MK2), as judged by the MTT assay. Cytotoxicity involving necrosis and apoptosis has been described in the Cells of both lines were exposed to a concentration of venom cor- same cell lines with the venoms of Bothrops sp (Morais et al., 2013; responding to one CC50 for 24 h, after which cells were stained with Mello et al., 2014; Marinho et al., 2015). It was previously shown that annexin (to assess apoptosis) and with 7AAD (to assess plasma mem- the venoms of H. hypnale, H. zara and H. nepa inhibited cell prolifera- brane disruption, i.e., necrosis). As depicted in Fig. 2, necrosis was the tion of rat aorta smooth muscle cells at a relatively low concentration predominant effect at the venom concentration tested. (Maduwage et al., 2011). In our observations, when cells were in-

Cells of both lines exposed to a concentration of venom corre- cubated with a venom concentration corresponding to one CC50, for sponding to one and two CC50 were stained with Rhodamine, in order 24 h, more cells were positively stained with 7AAD than with annexin, to assess the effects on the mitochondrial membrane potential. Venom thus indicating that at this dose and time of incubation the majority of induced a drastic reduction in Rhodamine fluorescence at both con- cells was affected by necrosis instead of apoptosis. It has to be con- centrations (Fig. 3), hence evidencing a drop in the mitochondrial sidered, however, that the balance between necrosis and apoptosis in membrane potential. Moreover, venom induced an increment in the this model may be shifted when using other venom concentrations, as concentration of reactive oxygen species in the two kidney-derived cells shown for a lymphoblastoid cell line incubated with a myotoxic PLA2 lines, at concentrations corresponding to one and two CC50, as revealed homologue from the venom of Bothrops asper (Mora et al., 2005). The by an increment in the relative fluorescence of DCFH-DA (Fig. 4). direct toxicity on tubular epithelial cell lines by necrosis can lead to a

42 M. Villalta, et al. Toxicon 165 (2019) 40–46

Fig. 1. Effects of H. hypnale venom (10 μg/mL), on perfusion pressure (A), renal vascular resistance (B), urinary flow (C), glomerular filtration rate (D), percent of sodium, potassium and chloride tubular transport (E, G, I) and percent of proximal tubule sodium, potassium and chloride transport (F, H, J), in the isolated perfused rat kidneys. The columns and bars represent the mean ± S.E.M. for five rats. The results in samples treated with venom were compared to the control corresponding to the first 30 min of perfusion with Krebs-Henseleit solution before venom was added (black bar) (*p < 0.05 as compared to control).

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Fig. 2. Effects on cell death of the H. hypnale venom measured by Annexin V-PE and 7-AAD staining and detected by flow cytometry. MDCK (B, C) and LLC-MK2 (E,

F) cells were treated with 1 CC50 of H. hypnale venom for 24 h. The control group corresponds to cells without venom treatment (A: MDCK, D: LLC-MK2). On the flow cytometric scatter graphs, the left lower quadrant represents the live cells, the right lower quadrant represents the population of early apoptotic cells, the right upper quadrant represents the accumulation of cells in terminal stage of cell death, and the left upper quadrant corresponds to the population of necrotic cells. Results in C and F correspond to means ± S.E.M. of three replicates from three independent experiments (*p < 0.05 as compared to controls).

Fig. 3. H. hypnale venom effects on mitochondrial transmembrane potential. MDCK (A, B) and LLC-MK2 (C, D) cells were treated with 1 CC50 and 2 CC50 of H. hypnale venom for 24 h. Then, cells were stained with rhodamine 123 (Rho123) and analyzed in the flow cytometer to measure the decrease in Rho123 accumulation in mitochondria. A and C show a histogram of representative mitochondrial transmembrane potential analysis in cell populations treated with venom. B and D present the fluorescence ratio relative to control ± SEM, of three replicates from three independent experiments (*p < 0.05 as compared to control).

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Fig. 4. Intracellular ROS generation induced by H. hypnale venom (1 CC50 and 2 CC50) in MDCK (A, B) and LLC-MK2 (C, D) cells. After treatment with venom for 24 h, cells were incubated with 5 mM DCFH-DA for 30 min and then immediately submitted to flow cytometric analysis. ROS generation was expressed as a ratio of relative fluorescent intensity compared to the control group. A representative histogram of three independent experiments for each sample is presented (A, C). Generation of ROS in the cytoplasm is demonstrated by an increment in fluorescence. Summarized data show intracellular ROS content, expressed as the ratio of mean fluorescence of treated groups relative to that of untreated control (B, D). Data are means ± S.E.M. of three replicates from three independent experiments and compared with sample without any treatment (*p < 0.05 as compared to controls). rapid cell loss in vivo, with a consequent electrolyte dysfunction, as a assess whether this antivenom is effective at abrogating the renal effects contributory factor to the development of AKI. of the venom. Experiments were also performed with Rhodamine 123 in order to In conclusion, our findings provide evidence of a nephrotoxic action assess the effect on the mitochondrial membrane potential and DCF to of the venom of H. hypnale, as demonstrated by perturbation of several assess the ROS production. There was a drastic reduction in this po- renal functions in an isolated kidney preparation and by a direct cy- tential and an increase on ROS generation, underscoring a process of totoxic effect on proximal and distal tubular epithelial cell lines. These cell damage. The loss of mitochondrial membrane potential has been experimental observations are in line with previous experimental stu- associated with the opening of the permeability transition pore in mi- dies describing nephrotoxicity by this venom, and by clinical reports tochondrial membranes (Bernardi and Rasola, 2007), a mega-channel highlighting the potential of H. hypnale venom to induce AKI. The that can assemble as a consequence of mitochondrial calcium overload, mechanisms of this pathophysiological effect and its neutralization by owing to increments in cytosolic calcium concentration following antivenoms deserve further investigations. plasma membrane damage, and also following an increment in reactive oxygen species in the cell (Rottenberg and Hoek, 2017). It is therefore Declaration of interests suggested that cytotoxicity induced by H. hypnale venom in these cell lines is likely to depend on a direct action of venom cytotoxic compo- The authors declare that they have no known competing financial nents on the plasma membrane, with a disruption in its integrity. This is interests or personal relationships that could have appeared to influ- fl ff followed by a calcium in ux which directly a ects mitochondria and ence the work reported in this paper. contributes to the generation of reactive oxygen species, ending in ir- reversible cell damage. Owing to the high proportion of phospholipases Ethical statement A2 in the proteome of this venom (Ali et al., 2013; Tan et al., 2015; Vanuopadath et al., 2018), it is proposed that venom cytotoxicity is due The experimental protocol used with rats was approved the Ethics to the action of this hydrolytic enzymes on plasma membrane phos- Committee on Animal Research (CEPA) of the Federal University of pholipids, a hypothesis that awaits the isolation of these components. Ceara (Permit Number 79/08). The antivenoms used in Sri Lanka and the Indian peninsula do not include Hypnale sp venom in their immunizing mixture, they are in- effective in envenomings by H. hypnale (Sellahewa et al., 1995; Keyler Acknowledgements et al., 2013). Recently, a new polyspecific antivenom was developed which included the venom of H. hypnale in the immunizing scheme. This study was supported by Vicerrectoría de Investigación, This antivenom proved effective in the neutralization of lethal, he- Universidad de Costa Rica (project No. 741-B4-102), and by the Graduate Studies System of this university for supporting a research morrhagic, in vitro coagulant, proteinase and phospholipase A2 activ- ities of H. hypnale venom (Villalta et al., 2016). It would be relevant to stay of M. Villalta at the Federal University of Ceará. Thanks are due to Daniel Keyler for fruitful discussions. This work was carried out by

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