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Disarray of glomerular and tubular cell adhesion molecules in the course of experimental Bothrops moojeni envenomation
Toxicon 78 (2014) 41–46
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Disarray of glomerular and tubular cell adhesion molecules in the course of experimental Bothrops moojeni envenomation Carla Beatriz Collares-Buzato 1, Maria Alice da Cruz-Höfling*,1 Department of Histology and Embryology, Institute of Biology, P.O. Box 6109, State University of Campinas (UNICAMP), 13 087-130 Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2013 Received in revised form 11 November 2013 Accepted 20 November 2013 Available online 27 November 2013
In this study, we show that administration of Bothrops moojeni venom in rats induces a general disturbance in the distribution and content of the tight junctional protein ZO-1, the cell-matrix receptor beta 1 integrin, the cytoskeletal proteins, vinculin and F-actin, and of the extracellular matrix component laminin in renal corpuscles and cortical nephron tubules. These findings suggest that cell-cell and cell-matrix adhesion proteins may be molecular targets in the B. moojeni-induced kidney injury. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acute kidney injury Bothropic envenomation Cell adhesion proteins Cytoskeletal proteins Glomerular injury Nephron tubular injury
The most serious systemic effect and common complication in lethal cases of Bothrops snakebite accidents is the acute kidney injury (AKI) associated to acute injury to renal tubules and glomeruli (Boer-Lima et al., 1999, 2002; Linardi et al., 2011; Sgrignolli et al., 2011). The molecular mechanisms underlying the AKI after Bothrops envenoming are not completely understood but may be a result of haemodynamic alterations and direct toxic effects in renal parenchyma induced by venom components (Boer-Lima et al., 1999, 2002; Burdmann et al., 1993; Collares-Buzato et al., 2002; Nascimento et al., 2007). In vivo and in vitro studies suggest that the nephrotoxic injury induced by Bothrops moojeni venom may involve disruption of normal cell-cell and cell-matrix interactions and alterations to the membrane permeability and epithelial polarity in various regions of the renal tubules. Nephrotoxic effects assessed by systemic B. moojeni venom administration in rats showed podocyte foot processes * Corresponding author. Tel.: þ55 19 3521 6224. E-mail address: hofl[email protected] (M.A.da Cruz-Höfling). 1 The authors contributed equally to the study. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.11.013
effacement, mesangiolysis, microaneurysms and visceral basement membrane disturbances as well as tubule necrosis preceded by cell-to-cell detachment (Boer-Lima et al., 1999, 2002). Exposure of the renal tubular MDCK cell line to B. moojeni crude venom resulted in disruption of the epithelial barrier function associated with detachment of the monolayer from the substratum, and alterations to the distribution of the junctional proteins, occludin, ZO-1, and E-cadherin (Collares-Buzato et al., 2002). Moreover, we reported a disarray of the cell cytoskeleton, specifically of stress fibres associated to focal adhesions at the cellmatrix contacts after MDCK venom exposure (CollaresBuzato et al., 2002). The relevance of studying the molecular mechanisms involved in renal disturbances caused by ophidism is obvious; it not only can give a better understanding of how to counteract the venom effects but also contribute to our knowledge of kidney pathophysiology. In this study, we investigated whether B. moojeni snake venom-induced nephrotoxicity in rats is associated with changes in the cell content and distribution of some adhesion proteins in kidney parenchyma.
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C.B. Collares-Buzato, M.A.da Cruz-Höfling / Toxicon 78 (2014) 41–46
Lyophilized B. moojeni snake venom from a pool of harvested samples was donated by the Instituto Butantan (São Paulo, SP, Brazil) and stored at 20 C. Male Wistar rats (200–300 g) were obtained from an established colony maintained by the University’s Central Animal House Service (CEMIB/UNICAMP). All experimental protocols were approved by the institutional Committee for Ethics in Animal Use (CEUA/IB, UNICAMP, protocol 1990-1). The rats were anesthetized with an i.p. injection (2 mg/mg body mass) of 3:1 ketamine chloride (Dopalen, 100 mg/kg) and xylazine chloride (Anasedan, 10 mg/kg) (Vetbrands, Jacarei, SP, Brazil) and then received an intravenous injection (0.5 ml) of freshly prepared 0.15 M NaCl (control group) or 0.4 mg/kg B. moojeni venom solution (treated group) in the tail vein (n ¼ 18 animals/group). The dose of 0.4 mg/kg was previously used in in vivo studies (Boer-Lima et al., 1999, 2002) and chosen based on an estimative of the venom volume inoculated in a human snakebite (Burdmann et al., 1993). After saline or venom injection, the rats were allowed to survive for 5, 16 and 48 h (n ¼ 6 animals/time) after which they were euthanized with an overdose of anesthetics. Then, the kidneys were removed, cut in half and frozen in n-hexane with liquid N2 and stored at 70 C until use. Cryosections (5 mm thick), obtained in a cryostat adjusted at 20 C, were transferred to poly-L-lysine-coated glass slides. After 3 min-fixation with 20 C acetone, the kidney sections were processed for indirect immunofluorescence technique (Peixoto and Collares-Buzato, 2006) using the following primary antibodies all at 1:50 dilution: rabbit anti-ZO-1 (Zymed Laboratories, San Francisco, CA, USA), mouse anti-vinculin (Sigma, St. Louis, MO, USA), mouse anti-laminin (Sigma) and mouse anti-integrin beta 1 (Sigma). After incubation with the specific secondary antibody conjugated with fluorescein (dilution 1:75), the sections were mounted in a commercial anti-fading agent (Vectashield, Vector Laboratories, Burlingame, CA, USA). Cytoskeletal F-actin was labelled in 3.7% formalin-fixed kidney cryosections with TRITC-conjugated phalloidin, as previously described (Damico et al., 2007). All cell labellings were visualised and images captured by confocal laser scanning microscopy (CLSM; BioRad MRC 1024UV; BioRad, Hercules, CA, USA) using an inverted fluorescence microscope. To permit comparison between the venomtreated and control groups, the tissue staining and microscopic examination of all groups were performed in the same experimental session using the same confocal parameters. The glomerular fluorescence intensity for ZO-1, vinculin and integrin beta 1 was measured in all images obtained using the free software Image J (http://rsbweb. nih.gov/ij/) and expressed as integrated density per renal corpuscle area. All numerical results were expressed as means standard error of the mean (SEM). For multiple comparisons, statistical significance was assessed by Oneway ANOVA followed by the Bonferroni’s post-test. For comparison between groups, statistical significance was assessed using Student’s t test (two tailed). The significance level was set at P 0.05. All statistical analyses were performed using the GraphPad Prism Version 5.00 for Windows (GraphPad Software, La Jolla, USA).
Figs. 1 and 2 show the alterations to the tissue content and distribution of ZO-1, beta 1 integrin, vinculin, F-actin and laminin in renal corpuscles and cortical nephron tubular cells, respectively, after B. moojeni venom administration. In normal kidneys from Wistar control rats, the immunofluorescence labelling for ZO-1, a tight junctionassociated protein, was uniformly distributed along the capillary loops, as expected from the distribution of the glomerular slit diaphragms (Kurihara et al., 1992), in all glomeruli examined and endothelial wall of the afferent and efferent arterioles (Fig. 1A), and at the apical junctional complexes of tubular cells (Fig. 2A). In envenomed rats, the immunoreaction for this intercellular junctional protein was significantly reduced in both glomeruli and afferent and efferent arterioles (Fig. 1B–D) and in some cortical nephron tubules (Fig. 2B). The lowest value of glomerular ZO-1 immunofluorescence was detected at 16 h after envenoming, with no complete recovery to the control values even after 48 h of venom administration (Fig. 1E). In podocytes, ZO-1 is known to anchor the slit diaphragm proteins to the actin cytoskeleton, therefore it plays a role to keep the structure and functionality of the filtration slits (Schnabel et al., 1990). Alterations to the glomerular distribution and/or decrease in podocyte content of ZO-1 have been implicated in the pathogenesis of proteinuria in animal models (Kurihara et al., 1992; Macconi et al., 2000; Rincón-Choles et al., 2006) and in human AKI (Krautkramer et al., 2011). In addition, disruption of the tight junction-associated ZO-1 distribution in tubular cells is well-known to result in impairment of the epithelial barrier (Collares-Buzato et al., 1998), and can be associated with acute tubular injury (Krautkramer et al., 2011). Since B. moojeni venom seems to induce impairment of cell-matrix interaction in renal parenchyma (Boer-Lima et al., 1999, 2002) and cultured tubular cells (CollaresBuzato et al., 2002), we went to see the distribution of beta 1 integrin in kidney cryosections of envenomed rats. Integrins are heterodimeric, noncovalently associated glycoprotein complexes consisting of an alpha and a beta chain, and functions as a cell membrane receptor that enable cells to adhere to matrix compounds. The beta 1 integrins are the largest group, composed of a beta 1 chain associated with 1 of 12 alpha chains, that have been shown to be expressed in the kidney (Kagami and Kondo, 2004; Molina et al., 2005). In our control rats, beta 1 integrin immunoreactivity was observed at the intra-glomerular mesangial areas of the renal corpuscle, as faint lines around the glomeruli capillaries (in cross-sections) (Fig. 1F), and at the basal region of the plasma membrane of tubular cells (Fig. 2C). This beta 1 integrin distribution is very similar to that described previously by others, in rodent and human kidney (Hillis et al., 1997; Kagami and Kondo, 2004; Molina et al., 2005). Subtle changes in the glomeruli immunolabelling for this cell-matrix receptor were seen after envenoming characterized mainly by a more diffuse labelling of the glomeruli capillaries (Fig. 1G– I), with no significant changes in the immunofluorescence intensity (Fig. 1J). Meanwhile, venom administration induced a detectable decrease of integrin associated to the tubular basal membrane in some areas of the cortical renal
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Fig. 1. B. moojeni venom induced changes in content and distribution of matrix- and cell adhesion-associated proteins in renal corpuscles of rats. Panels show cryosections of representative renal corpuscles labelled for ZO-1 (A–D), beta 1 integrin (F–I), vinculin (K–N), F-actin (P–S) and laminin (T–X) in control animals (A,F,K,P,T) and in treated animals after 5 h (B,G,L,Q,U), 16 h (C,H,M,R,V), and 48 h (D,I,N,S,X) of venom administration. ZO-1, integrin, vinculin and laminin were detected by indirect immunofluorescence. F-actin was localized by phalloidin reaction. Graphs in E, J and O show the measurement of fluorescence degree of ZO-1 (E), integrin (J) and vinculin (O) glomerular immunoreactions normalised by glomerular area and expressed as means SEM (from 2 to 4 independent immunoreactions/protein). Scale bars ¼ 50 mm (bar in image D is valid for A to D and T to X; bar in image I is valid for F to I, K to N and P to S). **P < 0.01; ***P < 0.001 in comparison with control levels (One-way ANOVA followed by Bonferroni’s post-test).
parenchyma, especially at 16 h post-venom (Fig. 2D). In an in vivo model of AKI induced by ischemia, it has been proposed that a decrease in beta 1 integrin on basal surfaces results in tubular epithelia detachment into lumen, leading to tubular obstruction and back leak of glomerular filtrate (Molina et al., 2005). Therefore, it is plausible to suggest that the decrease in beta 1 integrin tubular content contributes to the disruption in renal tubular structure and function seen after Bothrop moojeni venom (Boer-Lima et al., 1999).
Vinculin, a cytoskeleton-associated protein, was detected by immunofluorescence in renal corpuscles of salineinjected control rats around the capillary loops, at the parietal epithelium and mainly in mesangial cells (Fig. 1K). After 5 h and 16 h of venom injection, the immunolabelling for vinculin was significantly increased at the glomerulus as a whole (Fig. 1L–N), being difficult to make the differentiation among the corpuscle cells (mesangial, endothelial cells and podocytes). The increase in vinculin immunoreaction was confirmed quantitatively, by the
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Fig. 2. B. moojeni venom induced a decrease in cortical nephron tubular content of matrix- and cell adhesion-associated proteins in rats. Panels show representative cryosections of nephron tubules labelled for ZO-1 (A,B), beta 1 integrin (C,D), vinculin (E,F), F-actin (G,H) and laminin (I,J) in control animals (A,C,E,G,I) and in treated animals after 16 h (B,D,F,H,J) of venom administration. ZO-1, integrin, vinculin and laminin were detected by indirect immunofluorescence (in 2–4 independent immunoreactions/protein). F-actin was localized by phalloidin reaction (in 4 independent experiments). Scale bar ¼ 50 mm.
measurement of glomeruli fluorescence intensity (Fig. 1O). In tubular epithelia of control animals, vinculin immunoreaction was detected as straight lines associated to the basal plasma membrane (Fig. 2E). This tubular distribution of vinculin subtly changed after B. moojeni envenoming, appearing more discontinuous (arrow in Fig. 2F), which may suggest a decrease in the association of vinculin with focal contacts at the basal membrane of tubular cells after treatment. This finding is in accordance with previous works showing that Bothrops venoms can induce impairment of the cell-matrix interaction in cultured renal tubular epithelia (Collares-Buzato et al., 2002; Nascimento
et al., 2007). Mesangial cells, that highly express vinculin (Berfield et al., 1997; Hartner et al., 1999), are shown to proliferate as response to acute or repetitive glomerular injury (Hartner et al., 1999), including that induced by Habu snake venom (Cattell, 1979). This may explain, at least in part, the increases in the glomerular immunoreaction to vinculin after B. moojeni envenomation observed herein, therefore suggesting a possible compensatory expansion of mesangial cells after venom-induced glomerulonephritis. Nevertheless, this explanation may be unlikely if ones consider the timetable of the appearance of this alteration (as early as 5 h after venom administration) and the fact that no mesangial proliferation (but mesangiolysis) was reported after ultrastructural analysis of renal corpuscles in envenomed rats (Boer-Lima et al., 2002). Alternatively, the increased glomerular immunofluorescence for vinculin may be a result of an increased expression of this cytoskeletal protein on podocytes, similarly to that reported in experimental and clinical cases of proteinuria associated with podocyte foot process effacement (Miao et al., 2009), and in agreement with glomerular filtration dysfunction reported by Boer-Lima and co-workers in rats envenomed with B. moojeni (1999, 2002). The cytoskeletal protein F-actin, labelled with phalloidin, was detected in all cells of the renal corpuscle: in podocyte foot processes around capillary loops, in mesangial cells and in the parietal epithelium (Fig. 1P). In cortical tubules, F-actin was mainly found associated to the basal plasma membrane in all renal tubules and to the apical brush border of proximal tubules (Fig. 2G). B. moojeni venom induced minor changes in both F-actin content and distribution in the renal corpuscles (Fig. 1Q–S) but led to marked decrease in the labelling of the brush border and basal region of the tubular epithelia (Fig. 2H), which are in accordance with our previous histological findings (BoerLima et al., 1999). Since labelling of F-actin with phalloidin permitted the observation of all components of the renal corpuscles, this reaction revealed several morphological abnormalities after venom administration, already reported (Boer-Lima et al., 2002), such as capillary microaneurisms (arrow in Fig. 1Q), over-sized capsular space, and glomeruli tuft lobulation and retraction (asterisk in Fig. 1R). The frequency of these venom-induced alterations to the renal corpuscle morphology and tubular F-actin labelling was markedly higher at 16 h after venom administration (92.3% of all corpuscles and 100% of all cortical tubules observed) in comparison with the other time points analysed (5 h, 65% of all corpuscles and 41.7% of all cortical tubules analysed; 48 h, 70% of all corpuscles and 50% of all cortical tubules analysed). In control rats, the extracellular matrix protein laminin was detected as a linear labelling along the glomerular capillary wall, as well as along the basement membrane of both Bowman’s capsule parietal and tubular epithelia (Figs. 1T and 2I, respectively). In envenomed rats, the laminin immunostaining was more diffuse, with a tape-like appearance in glomeruli and parietal leaflet of the renal corpuscle (Fig. 1U–X). In addition, the immunoreaction for laminin appeared discontinuous at the tubular basement membrane of a number of cortical nephron tubules after envenoming (Fig. 2J). Boer-Lima et al. (2002) have
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described structural disturbances of the glomerular basement membrane (GBM) in venom-treated rats, which is confirmed here with the labelling of one of the major matrix constituent of GBM, the laminin. As suggested previously, it is possible that laminin is one of the targets of proteolytic enzymes contained in the B. moojeni crude venom (Boer-Lima et al., 2002). In conclusion, we have presently described several alterations to the native expression and localization of some cell adhesion proteins and matrix-associated proteins that may be involved in the genesis of the acute kidney injury and the severe proteinuria induced by B. moojeni venom. In the renal corpuscle, the decreases in glomerular content of ZO-1, the disorganization of laminin within the GBM and the increases in vinculin podocyte expression are in accordance with the impairment of the glomerular filtration barrier seen after B. moojeni envenoming (Boer-Lima et al., 2002). In renal tubules, we observed a reduction in ZO-1 tight junctional level and in focal adhesion content of beta 1 integrin, vinculin and F-actin, associated with a disruption of brush border-bound F-actin and basement membrane laminin structure. All these alterations are compatible with the severe renal tubule disturbances induced by B. moojeni venom, which included tubular cell detachment associated with tubular necrosis and loss of proximal brush border leading to a sustained increase in tubular sodium rejection (Boer-Lima et al., 1999). Therefore, cell-cell and cell-matrix adhesion proteins seems to be molecular targets in the B. moojeni venom-induced kidney injury, being result of direct action of venom compounds (metalloproteinases, desintegrins, and phospholipase A2) (Assakura et al., 1985; Calgarotto et al., 2008; Cruz-Höfling et al., 2001; Barbosa et al., 2002; Demler et al., 2010; Serrano et al., 1993; Queiroz et al., 2008) and/or consequence of venom-induced renal ischemia (Boer-Lima et al., 1999, 2002; Burdmann et al., 1993). Acknowledgements The authors thank Instituto Butantan (São Paulo, SP, BR) for venom donation. The authors also thank Ms. Christiane A.B. Tarsitano and Ms. Marta Beatriz Leonardo for excellent technical assistance. This work has been funded by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP # 98/341-0). M.A.C.H. (CNPq # 302206/ 2008-6) and CBC-B (CNPq # 307163/2012-1) are recipients of Research Fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). Conflict of interest None of the authors have any conflicts of interest related to this work. References Assakura, M.T., Reichl, A.P., Asperti, M.C.A., Mandelbaum, F.R., 1985. Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). Toxicon 23, 691–706. Barbosa, P.S., Havt, A., Facó, P.E., Sousa, T., Bezerra, I.S., Fonteles, M.C., Toyama, M.H., Marangoni, S., Novello, J.C., Monteiro, H.S., 2002. Renal
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Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Disarray of glomerular and tubular cell adhesion molecules in the course of experimental Bothrops moojeni envenomation Carla Beatriz Collares-Buzato 1, Maria Alice da Cruz-Höfling*,1 Department of Histology and Embryology, Institute of Biology, P.O. Box 6109, State University of Campinas (UNICAMP), 13 087-130 Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2013 Received in revised form 11 November 2013 Accepted 20 November 2013 Available online 27 November 2013
In this study, we show that administration of Bothrops moojeni venom in rats induces a general disturbance in the distribution and content of the tight junctional protein ZO-1, the cell-matrix receptor beta 1 integrin, the cytoskeletal proteins, vinculin and F-actin, and of the extracellular matrix component laminin in renal corpuscles and cortical nephron tubules. These findings suggest that cell-cell and cell-matrix adhesion proteins may be molecular targets in the B. moojeni-induced kidney injury. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acute kidney injury Bothropic envenomation Cell adhesion proteins Cytoskeletal proteins Glomerular injury Nephron tubular injury
The most serious systemic effect and common complication in lethal cases of Bothrops snakebite accidents is the acute kidney injury (AKI) associated to acute injury to renal tubules and glomeruli (Boer-Lima et al., 1999, 2002; Linardi et al., 2011; Sgrignolli et al., 2011). The molecular mechanisms underlying the AKI after Bothrops envenoming are not completely understood but may be a result of haemodynamic alterations and direct toxic effects in renal parenchyma induced by venom components (Boer-Lima et al., 1999, 2002; Burdmann et al., 1993; Collares-Buzato et al., 2002; Nascimento et al., 2007). In vivo and in vitro studies suggest that the nephrotoxic injury induced by Bothrops moojeni venom may involve disruption of normal cell-cell and cell-matrix interactions and alterations to the membrane permeability and epithelial polarity in various regions of the renal tubules. Nephrotoxic effects assessed by systemic B. moojeni venom administration in rats showed podocyte foot processes * Corresponding author. Tel.: þ55 19 3521 6224. E-mail address: hofl[email protected] (M.A.da Cruz-Höfling). 1 The authors contributed equally to the study. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.11.013
effacement, mesangiolysis, microaneurysms and visceral basement membrane disturbances as well as tubule necrosis preceded by cell-to-cell detachment (Boer-Lima et al., 1999, 2002). Exposure of the renal tubular MDCK cell line to B. moojeni crude venom resulted in disruption of the epithelial barrier function associated with detachment of the monolayer from the substratum, and alterations to the distribution of the junctional proteins, occludin, ZO-1, and E-cadherin (Collares-Buzato et al., 2002). Moreover, we reported a disarray of the cell cytoskeleton, specifically of stress fibres associated to focal adhesions at the cellmatrix contacts after MDCK venom exposure (CollaresBuzato et al., 2002). The relevance of studying the molecular mechanisms involved in renal disturbances caused by ophidism is obvious; it not only can give a better understanding of how to counteract the venom effects but also contribute to our knowledge of kidney pathophysiology. In this study, we investigated whether B. moojeni snake venom-induced nephrotoxicity in rats is associated with changes in the cell content and distribution of some adhesion proteins in kidney parenchyma.
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Lyophilized B. moojeni snake venom from a pool of harvested samples was donated by the Instituto Butantan (São Paulo, SP, Brazil) and stored at 20 C. Male Wistar rats (200–300 g) were obtained from an established colony maintained by the University’s Central Animal House Service (CEMIB/UNICAMP). All experimental protocols were approved by the institutional Committee for Ethics in Animal Use (CEUA/IB, UNICAMP, protocol 1990-1). The rats were anesthetized with an i.p. injection (2 mg/mg body mass) of 3:1 ketamine chloride (Dopalen, 100 mg/kg) and xylazine chloride (Anasedan, 10 mg/kg) (Vetbrands, Jacarei, SP, Brazil) and then received an intravenous injection (0.5 ml) of freshly prepared 0.15 M NaCl (control group) or 0.4 mg/kg B. moojeni venom solution (treated group) in the tail vein (n ¼ 18 animals/group). The dose of 0.4 mg/kg was previously used in in vivo studies (Boer-Lima et al., 1999, 2002) and chosen based on an estimative of the venom volume inoculated in a human snakebite (Burdmann et al., 1993). After saline or venom injection, the rats were allowed to survive for 5, 16 and 48 h (n ¼ 6 animals/time) after which they were euthanized with an overdose of anesthetics. Then, the kidneys were removed, cut in half and frozen in n-hexane with liquid N2 and stored at 70 C until use. Cryosections (5 mm thick), obtained in a cryostat adjusted at 20 C, were transferred to poly-L-lysine-coated glass slides. After 3 min-fixation with 20 C acetone, the kidney sections were processed for indirect immunofluorescence technique (Peixoto and Collares-Buzato, 2006) using the following primary antibodies all at 1:50 dilution: rabbit anti-ZO-1 (Zymed Laboratories, San Francisco, CA, USA), mouse anti-vinculin (Sigma, St. Louis, MO, USA), mouse anti-laminin (Sigma) and mouse anti-integrin beta 1 (Sigma). After incubation with the specific secondary antibody conjugated with fluorescein (dilution 1:75), the sections were mounted in a commercial anti-fading agent (Vectashield, Vector Laboratories, Burlingame, CA, USA). Cytoskeletal F-actin was labelled in 3.7% formalin-fixed kidney cryosections with TRITC-conjugated phalloidin, as previously described (Damico et al., 2007). All cell labellings were visualised and images captured by confocal laser scanning microscopy (CLSM; BioRad MRC 1024UV; BioRad, Hercules, CA, USA) using an inverted fluorescence microscope. To permit comparison between the venomtreated and control groups, the tissue staining and microscopic examination of all groups were performed in the same experimental session using the same confocal parameters. The glomerular fluorescence intensity for ZO-1, vinculin and integrin beta 1 was measured in all images obtained using the free software Image J (http://rsbweb. nih.gov/ij/) and expressed as integrated density per renal corpuscle area. All numerical results were expressed as means standard error of the mean (SEM). For multiple comparisons, statistical significance was assessed by Oneway ANOVA followed by the Bonferroni’s post-test. For comparison between groups, statistical significance was assessed using Student’s t test (two tailed). The significance level was set at P 0.05. All statistical analyses were performed using the GraphPad Prism Version 5.00 for Windows (GraphPad Software, La Jolla, USA).
Figs. 1 and 2 show the alterations to the tissue content and distribution of ZO-1, beta 1 integrin, vinculin, F-actin and laminin in renal corpuscles and cortical nephron tubular cells, respectively, after B. moojeni venom administration. In normal kidneys from Wistar control rats, the immunofluorescence labelling for ZO-1, a tight junctionassociated protein, was uniformly distributed along the capillary loops, as expected from the distribution of the glomerular slit diaphragms (Kurihara et al., 1992), in all glomeruli examined and endothelial wall of the afferent and efferent arterioles (Fig. 1A), and at the apical junctional complexes of tubular cells (Fig. 2A). In envenomed rats, the immunoreaction for this intercellular junctional protein was significantly reduced in both glomeruli and afferent and efferent arterioles (Fig. 1B–D) and in some cortical nephron tubules (Fig. 2B). The lowest value of glomerular ZO-1 immunofluorescence was detected at 16 h after envenoming, with no complete recovery to the control values even after 48 h of venom administration (Fig. 1E). In podocytes, ZO-1 is known to anchor the slit diaphragm proteins to the actin cytoskeleton, therefore it plays a role to keep the structure and functionality of the filtration slits (Schnabel et al., 1990). Alterations to the glomerular distribution and/or decrease in podocyte content of ZO-1 have been implicated in the pathogenesis of proteinuria in animal models (Kurihara et al., 1992; Macconi et al., 2000; Rincón-Choles et al., 2006) and in human AKI (Krautkramer et al., 2011). In addition, disruption of the tight junction-associated ZO-1 distribution in tubular cells is well-known to result in impairment of the epithelial barrier (Collares-Buzato et al., 1998), and can be associated with acute tubular injury (Krautkramer et al., 2011). Since B. moojeni venom seems to induce impairment of cell-matrix interaction in renal parenchyma (Boer-Lima et al., 1999, 2002) and cultured tubular cells (CollaresBuzato et al., 2002), we went to see the distribution of beta 1 integrin in kidney cryosections of envenomed rats. Integrins are heterodimeric, noncovalently associated glycoprotein complexes consisting of an alpha and a beta chain, and functions as a cell membrane receptor that enable cells to adhere to matrix compounds. The beta 1 integrins are the largest group, composed of a beta 1 chain associated with 1 of 12 alpha chains, that have been shown to be expressed in the kidney (Kagami and Kondo, 2004; Molina et al., 2005). In our control rats, beta 1 integrin immunoreactivity was observed at the intra-glomerular mesangial areas of the renal corpuscle, as faint lines around the glomeruli capillaries (in cross-sections) (Fig. 1F), and at the basal region of the plasma membrane of tubular cells (Fig. 2C). This beta 1 integrin distribution is very similar to that described previously by others, in rodent and human kidney (Hillis et al., 1997; Kagami and Kondo, 2004; Molina et al., 2005). Subtle changes in the glomeruli immunolabelling for this cell-matrix receptor were seen after envenoming characterized mainly by a more diffuse labelling of the glomeruli capillaries (Fig. 1G– I), with no significant changes in the immunofluorescence intensity (Fig. 1J). Meanwhile, venom administration induced a detectable decrease of integrin associated to the tubular basal membrane in some areas of the cortical renal
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Fig. 1. B. moojeni venom induced changes in content and distribution of matrix- and cell adhesion-associated proteins in renal corpuscles of rats. Panels show cryosections of representative renal corpuscles labelled for ZO-1 (A–D), beta 1 integrin (F–I), vinculin (K–N), F-actin (P–S) and laminin (T–X) in control animals (A,F,K,P,T) and in treated animals after 5 h (B,G,L,Q,U), 16 h (C,H,M,R,V), and 48 h (D,I,N,S,X) of venom administration. ZO-1, integrin, vinculin and laminin were detected by indirect immunofluorescence. F-actin was localized by phalloidin reaction. Graphs in E, J and O show the measurement of fluorescence degree of ZO-1 (E), integrin (J) and vinculin (O) glomerular immunoreactions normalised by glomerular area and expressed as means SEM (from 2 to 4 independent immunoreactions/protein). Scale bars ¼ 50 mm (bar in image D is valid for A to D and T to X; bar in image I is valid for F to I, K to N and P to S). **P < 0.01; ***P < 0.001 in comparison with control levels (One-way ANOVA followed by Bonferroni’s post-test).
parenchyma, especially at 16 h post-venom (Fig. 2D). In an in vivo model of AKI induced by ischemia, it has been proposed that a decrease in beta 1 integrin on basal surfaces results in tubular epithelia detachment into lumen, leading to tubular obstruction and back leak of glomerular filtrate (Molina et al., 2005). Therefore, it is plausible to suggest that the decrease in beta 1 integrin tubular content contributes to the disruption in renal tubular structure and function seen after Bothrop moojeni venom (Boer-Lima et al., 1999).
Vinculin, a cytoskeleton-associated protein, was detected by immunofluorescence in renal corpuscles of salineinjected control rats around the capillary loops, at the parietal epithelium and mainly in mesangial cells (Fig. 1K). After 5 h and 16 h of venom injection, the immunolabelling for vinculin was significantly increased at the glomerulus as a whole (Fig. 1L–N), being difficult to make the differentiation among the corpuscle cells (mesangial, endothelial cells and podocytes). The increase in vinculin immunoreaction was confirmed quantitatively, by the
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Fig. 2. B. moojeni venom induced a decrease in cortical nephron tubular content of matrix- and cell adhesion-associated proteins in rats. Panels show representative cryosections of nephron tubules labelled for ZO-1 (A,B), beta 1 integrin (C,D), vinculin (E,F), F-actin (G,H) and laminin (I,J) in control animals (A,C,E,G,I) and in treated animals after 16 h (B,D,F,H,J) of venom administration. ZO-1, integrin, vinculin and laminin were detected by indirect immunofluorescence (in 2–4 independent immunoreactions/protein). F-actin was localized by phalloidin reaction (in 4 independent experiments). Scale bar ¼ 50 mm.
measurement of glomeruli fluorescence intensity (Fig. 1O). In tubular epithelia of control animals, vinculin immunoreaction was detected as straight lines associated to the basal plasma membrane (Fig. 2E). This tubular distribution of vinculin subtly changed after B. moojeni envenoming, appearing more discontinuous (arrow in Fig. 2F), which may suggest a decrease in the association of vinculin with focal contacts at the basal membrane of tubular cells after treatment. This finding is in accordance with previous works showing that Bothrops venoms can induce impairment of the cell-matrix interaction in cultured renal tubular epithelia (Collares-Buzato et al., 2002; Nascimento
et al., 2007). Mesangial cells, that highly express vinculin (Berfield et al., 1997; Hartner et al., 1999), are shown to proliferate as response to acute or repetitive glomerular injury (Hartner et al., 1999), including that induced by Habu snake venom (Cattell, 1979). This may explain, at least in part, the increases in the glomerular immunoreaction to vinculin after B. moojeni envenomation observed herein, therefore suggesting a possible compensatory expansion of mesangial cells after venom-induced glomerulonephritis. Nevertheless, this explanation may be unlikely if ones consider the timetable of the appearance of this alteration (as early as 5 h after venom administration) and the fact that no mesangial proliferation (but mesangiolysis) was reported after ultrastructural analysis of renal corpuscles in envenomed rats (Boer-Lima et al., 2002). Alternatively, the increased glomerular immunofluorescence for vinculin may be a result of an increased expression of this cytoskeletal protein on podocytes, similarly to that reported in experimental and clinical cases of proteinuria associated with podocyte foot process effacement (Miao et al., 2009), and in agreement with glomerular filtration dysfunction reported by Boer-Lima and co-workers in rats envenomed with B. moojeni (1999, 2002). The cytoskeletal protein F-actin, labelled with phalloidin, was detected in all cells of the renal corpuscle: in podocyte foot processes around capillary loops, in mesangial cells and in the parietal epithelium (Fig. 1P). In cortical tubules, F-actin was mainly found associated to the basal plasma membrane in all renal tubules and to the apical brush border of proximal tubules (Fig. 2G). B. moojeni venom induced minor changes in both F-actin content and distribution in the renal corpuscles (Fig. 1Q–S) but led to marked decrease in the labelling of the brush border and basal region of the tubular epithelia (Fig. 2H), which are in accordance with our previous histological findings (BoerLima et al., 1999). Since labelling of F-actin with phalloidin permitted the observation of all components of the renal corpuscles, this reaction revealed several morphological abnormalities after venom administration, already reported (Boer-Lima et al., 2002), such as capillary microaneurisms (arrow in Fig. 1Q), over-sized capsular space, and glomeruli tuft lobulation and retraction (asterisk in Fig. 1R). The frequency of these venom-induced alterations to the renal corpuscle morphology and tubular F-actin labelling was markedly higher at 16 h after venom administration (92.3% of all corpuscles and 100% of all cortical tubules observed) in comparison with the other time points analysed (5 h, 65% of all corpuscles and 41.7% of all cortical tubules analysed; 48 h, 70% of all corpuscles and 50% of all cortical tubules analysed). In control rats, the extracellular matrix protein laminin was detected as a linear labelling along the glomerular capillary wall, as well as along the basement membrane of both Bowman’s capsule parietal and tubular epithelia (Figs. 1T and 2I, respectively). In envenomed rats, the laminin immunostaining was more diffuse, with a tape-like appearance in glomeruli and parietal leaflet of the renal corpuscle (Fig. 1U–X). In addition, the immunoreaction for laminin appeared discontinuous at the tubular basement membrane of a number of cortical nephron tubules after envenoming (Fig. 2J). Boer-Lima et al. (2002) have
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described structural disturbances of the glomerular basement membrane (GBM) in venom-treated rats, which is confirmed here with the labelling of one of the major matrix constituent of GBM, the laminin. As suggested previously, it is possible that laminin is one of the targets of proteolytic enzymes contained in the B. moojeni crude venom (Boer-Lima et al., 2002). In conclusion, we have presently described several alterations to the native expression and localization of some cell adhesion proteins and matrix-associated proteins that may be involved in the genesis of the acute kidney injury and the severe proteinuria induced by B. moojeni venom. In the renal corpuscle, the decreases in glomerular content of ZO-1, the disorganization of laminin within the GBM and the increases in vinculin podocyte expression are in accordance with the impairment of the glomerular filtration barrier seen after B. moojeni envenoming (Boer-Lima et al., 2002). In renal tubules, we observed a reduction in ZO-1 tight junctional level and in focal adhesion content of beta 1 integrin, vinculin and F-actin, associated with a disruption of brush border-bound F-actin and basement membrane laminin structure. All these alterations are compatible with the severe renal tubule disturbances induced by B. moojeni venom, which included tubular cell detachment associated with tubular necrosis and loss of proximal brush border leading to a sustained increase in tubular sodium rejection (Boer-Lima et al., 1999). Therefore, cell-cell and cell-matrix adhesion proteins seems to be molecular targets in the B. moojeni venom-induced kidney injury, being result of direct action of venom compounds (metalloproteinases, desintegrins, and phospholipase A2) (Assakura et al., 1985; Calgarotto et al., 2008; Cruz-Höfling et al., 2001; Barbosa et al., 2002; Demler et al., 2010; Serrano et al., 1993; Queiroz et al., 2008) and/or consequence of venom-induced renal ischemia (Boer-Lima et al., 1999, 2002; Burdmann et al., 1993). Acknowledgements The authors thank Instituto Butantan (São Paulo, SP, BR) for venom donation. The authors also thank Ms. Christiane A.B. Tarsitano and Ms. Marta Beatriz Leonardo for excellent technical assistance. This work has been funded by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP # 98/341-0). M.A.C.H. (CNPq # 302206/ 2008-6) and CBC-B (CNPq # 307163/2012-1) are recipients of Research Fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). Conflict of interest None of the authors have any conflicts of interest related to this work. References Assakura, M.T., Reichl, A.P., Asperti, M.C.A., Mandelbaum, F.R., 1985. Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). Toxicon 23, 691–706. Barbosa, P.S., Havt, A., Facó, P.E., Sousa, T., Bezerra, I.S., Fonteles, M.C., Toyama, M.H., Marangoni, S., Novello, J.C., Monteiro, H.S., 2002. Renal
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