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K+-ATPase
Biochimica et Biophysica Acta 1810 (2011) 895–906
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na +/K +-ATPase Alessandra Linardi a, b, Thomaz A.A. Rocha e Silva a, b, Elen H. Miyabara c, Carla F. Franco-Penteado d, Kiara C. Cardoso a, Patrícia A. Boer e, Anselmo S. Moriscot f, José A.R. Gontijo e, Paulo P. Joazeiro g, Carla B. Collares-Buzato g, Stephen Hyslop a,⁎ a
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil Departamento de Ciências Fisiológicas, Faculdade de Ciências Médicas da Santa Casa de São Paulo, 01221-020, São Paulo, SP, Brazil c Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), 05508-900, São Paulo, SP, Brazil d Hemocentro, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil e Núcleo de Medicina e Cirurgia Experimental, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil f Departamento de Biologia Celular e Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), 05508-900, São Paulo, SP, Brazil g Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 14 February 2011 Received in revised form 23 May 2011 Accepted 1 June 2011 Available online 24 June 2011 Keywords: Acute renal failure Bothrops alternatus Collagen deposition F-actin Na+/K+-ATPase Renal function
a b s t r a c t Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na +/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na +/K +-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na +/K +-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na +/K +-ATPase α1 subunit were increased 6 h post-venom, whereas Na +/K+-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na +/K +-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na +/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Envenoming by Bothrops snakes can cause acute renal failure [1–3], with an overall incidence ranging from b3% [4–6] to ~ 38% [7]. Acute renal failure is an important cause of death among patients bitten by Bothrops species (accounting for ~ 80% of deaths in some series) [7–10]. Clinically, venom-induced acute renal failure may be caused by hypovolemia or hypoperfusion of renal tissue, a direct nephrotoxic action of venom components, or disseminated intravas-
⁎ Corresponding author. Tel.: + 55 19 3521 9536; fax: + 55 19 3289 2968. E-mail address: [email protected] (S. Hyslop). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.006
cular coagulation. The resulting histological renal damage involves acute glomerulonephritis or acute tubular or cortical necrosis, while the functional alterations include metabolic acidosis, hyperkalemia, hematuria, reduced urine output (oliguria or anuria) and elevated serum urea and creatinine concentrations [3,7–9,11]. Experimentally, Bothrops venoms can damage renal glomeruli, proximal and distal tubules and the basement membrane, with proliferation of the mesangial matrix, glomerular congestion, brush border disorganization and fibrin deposition [12–16]. Studies using cultured Madin-Darby canine kidney (MDCK) cells (a renal epithelial cell line) have shown that Bothrops venoms are directly cytotoxic to renal cells by mechanisms that involve extensive disruption of the cytoskeleton and cell death by necrosis [17,18].
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In addition to morphological damage, Bothrops venoms also cause functional alterations. In rats, Bothrops jararaca venom causes acute renal failure, with a reduction in the glomerular filtration rate, osmotic clearance and renal plasma flow, and an increase in serum creatinine levels; there are variable effects on sodium excretion and mean arterial blood pressure [12,13]. Similar studies with Bothrops moojeni venom have also described glomerular damage, tubular degeneration and desquamation, hematuria and a reduction in glomerular filtration rate, with increments in urinary sodium excretion, but no changes in arterial blood pressure and no fibrin deposition in glomerular capillaries [14,15]. These studies indicate that acute renal failure caused by Bothrops venoms is characterized by impaired renal function that involves a reduction in glomerular filtration and tubular sodium reabsorption. Renal Na +/K +-ATPase plays a pivotal role in the active transport of certain solutes and maintenance of intracellular electrolyte homeostasis, with functional alterations in this pump leading to a reduction in glomerular filtration and tubular sodium reabsorption [19]. The localization of Na +/K +-ATPase in the basolateral membrane is regulated by direct interactions with membrane-associated cytoskeletal proteins, and this polar distribution of Na +/K +-ATPase is essential for efficient enzyme function and sodium reabsorption by renal tubular cells [20,21]. Since the accumulation of Bothrops venom in the kidney [22,23] may contribute to acute renal failure experimentally and clinically, and since renal Na +/K +-ATPase is important for maintaining intracellular electrolyte homeostasis, such as sodium reabsorption by renal tubular cells [20,21], in this study we investigated the morphological and functional renal alterations caused by B. alternatus venom in rats and the changes in renal Na +/K +-ATPase expression and activity. This is the first study to examine changes in renal Na +/K +-ATPase following the administration of Bothrops venom in vivo and to correlate these with venom-induced functional alterations. 2. Materials and methods 2.1. Reagents and venom Adenosine 5′-triphosphate (sodium salt), bovine serum albumin, goat anti-mouse FITC-conjugated secondary antibody, ouabain, paraformaldehyde and TRITC-conjugated phalloidin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse monoclonal antibody to the α1-subunit of Na +/K +-ATPase was purchased from Upstate (Lake Placid, NY, USA), glycine was from Amersham-GE Lifesciences (Piscataway, NJ, USA) and tissue freezing medium was from Leica (Nussloch, Germany). Commercial kits for creatinine quantification were from CELM (Barueri, SP, Brazil). Isoflurane was from Cristália (Itapira, SP, Brazil). The other reagents of analytical grade were obtained from the suppliers indicated in the appropriate sections below. Ninety-six well plates were from Corning (Corning, MA, USA). Bothrops alternatus venom obtained from adult snakes of both sexes was purchased from the Centro de Extração de Toxinas Animais (CETA, Morungaba, SP, Brazil) and was stored lyophilized at −20 °C. 2.2. Animals Male Wistar-Hanover rats (200–250 g) were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at UNICAMP and were housed 6 animals/cage at 23 °C on a 12 h light/ dark cycle, with free access to food and water. The experimental protocols were approved by an institutional Committee for Ethics in Animal Experimentation (CEEA/UNICAMP, protocol no. 681–1) and were done according to the general ethical guidelines for animal use
established by the Brazilian Society of Laboratory Animal Science (SBCAL) and EC Directive 86/609/EEC for Animal Experiments. 2.3. Venom dose and administration protocol A single venom dose of 0.8 mg/kg was used in this work based on preliminary experiments in which doses of 0.4 and 1.6 mg/kg were also tested. Rats injected with 0.4 mg/kg showed minimal histological alterations, whereas those receiving 1.6 mg/kg generally died within a few minutes or hours after venom administration — this short survival time precluded adequate assessment of changes in renal function. The dose of 0.8 mg/kg provided the best combination of renal damage (assessed histologically) in relation to survival time. This dose was similar to that used in other experimental studies, e.g., 0.4 mg/kg, i.v., for Bothrops jararaca [13] and Bothrops moojeni [14,15] and broadly agreed with the clinical setting. Since B. alternatus produces venom yields of 60–400 mg [24–27], the injection of all available venom in a bite would correspond to a theoretical dose of 0.86–5.71 mg/kg in a 70 kg human, which is probably greater than the true range of doses. With the injection of only 50% of the available venom, the dose in humans would be 0.43–2.85 mg/kg, a range that includes the dose of 0.8 mg/kg used here. Based on these considerations, the venom dose used here was not unrealistic and was probably very close to the clinical situation. Rats (n = 6/group) were injected with venom (0.8 mg/kg, i.v.) or 0.15 M NaCl (control) and 6, 24, 48 and 72 h and 7 days later they were anesthetized with isoflurane and killed by exsanguination, after which the kidneys were removed and immediately immersed in the appropriate fixative for further tissue processing or frozen in liquid nitrogen and stored at − 80 °C. 2.4. Histological analysis At the intervals indicated above, the kidneys were removed and cut sagitally prior to fixation in buffered 10% formol solution for 24 h, followed by dehydration in a graded ethanol series and embedding in Histosec (Merck, Rio de Janeiro, RJ, Brazil). Sections 5 μm thick were stained with periodic acid-Schiff or Sirius red reagent for examination by light microscopy and polarized light microscopy, respectively. Computer-assisted morphometric analysis was done using a Nikon Eclipse E800 microscope and Image ProPlus software. Glomerular damage was quantified by measuring the area (μm 2) after manual tracing of each Bowman's capsule and the corresponding glomerular tuft in 15–20 randomly selected renal corpuscles in one section from each of six rats per group. The capsular space was calculated as the difference between the glomerular tuft area and Bowman's capsule area [28]. 2.5. Renal function Rats were injected with B. alternatus venom (0.8 mg/kg, i.v.) or 0.15 M NaCl (control) and renal function was studied at the intervals mentioned above using a LiCl (60 mmol/100 g of body weight) tap water load given by gavage (2% of body weight), as described elsewhere [14]. At the end of the experiment, the rats were anesthetized and blood samples (obtained by cardiac puncture) and urine samples were collected for analysis (see below). Arterial blood pressure was measured by a non-invasive tail-cuff method [29] immediately before the rats were killed. Plasma and urinary sodium and potassium concentrations were measured by flame photometry (model B262; Micronal, São Paulo, SP, Brazil). Creatinine was determined by the alkaline picrate method [30] using a SpectraMax340 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA) and proteinuria was determined according to Bradford [31], with bovine serum albumin as the standard. Renal clearance (C) was calculated by a standard formula
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(C = UV/P) using the plasma creatinine and lithium levels for each period, where U = urinary concentration, V = urinary volume and P = plasma concentration. Creatinine clearance was used to estimate the glomerular filtration rate and lithium clearance (CLi +) was used to assess sodium proximal tubule output. Fractional sodium (FENa +) and potassium (FEK +) excretions were calculated as CNa +/CCr and CK +/CCr, respectively, where CNa + and CK + are the ion clearances and CCr is the creatinine clearance. Fractional proximal (FEPNa +) and post-proximal (FEPPNa +) sodium excretions were calculated as CLi +/ CCr × 100 and CNa +/CLi + × 100, respectively [14].
and processed through two more cycles of homogenization and centrifugation. The final pellet was discarded and the pooled supernatants were centrifuged at 10,000 ×g for 15 min. The new supernatant was discarded and the pellet was resuspended in 2 ml of the same homogenization solution, aliquoted and stored at −80 °C for enzyme activity. Protein concentrations were determined according to Bradford [31].
2.6. Tissue processing
Na +/K +-ATPase activity was assayed as described by Forbush [32]. For this assay, 100 μl of sample was added to 500 μl of buffer containing 60 mM Tris–HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 4 mM Na2ATP, 4 mM MgCl2 and 1 mM EDTA followed by incubation for 10 min at 37 °C. Na +/K +-ATPase activity was calculated as the difference between the inorganic phosphate (Pi) released in control tubes and that released in identical samples containing 1 mM ouabain. Inorganic phosphate was determined according to Baginski et al. [33], as modified by Ottolenghi [34] and Brotherus et al. [35]. The resulting
One kidney from each pair of kidneys stored at − 80 °C was used to assay Na +/K +-ATPase activity (Section 2.7) while the second kidney was used for RNA isolation and real time PCR (Section 2.8). For Na +/ K +-ATPase activity, the kidney was homogenized in 0.05 M Tris–HCl, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA, centrifuged (1000 ×g, 10 min, 4 °C), and the supernatant was collected and stored on ice. The pellet was resuspended in half the original volume
A
2.7. Na +/K +-ATPase assay
B
p
* d
D
C
d v
* p
m m md
E
p F
* m
d p
Fig. 1. Renal cortical sections stained with Sirius red. The sections were obtained from saline-treated (control) rats (A) and 6 h (B), 24 h (C), 48 h (D), 72 h (E) and 7 days (F) after the injection of B. alternatus venom. Note the hypertrophied renal corpuscles and nodules that form a densely clumped, strongly stained mesangial matrix (solid arrows) and widening of the capsular space (*) in venom-treated rats. Note also the distended distal (d) and proximal (p) tubules containing cellular debris and amorphic material, and the dilated peritubular capillaries (dashed arrows). m - microaneurysms, md - macula densa, v - vascular pole. Bar: 30 μm (for all panels).
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absorbance was measured at 705 nm and the activity was expressed as nmol of Pi released/mg of protein/min.
2.9. Fluorescence microscopy of the α1 subunit of Na +/K +-ATPase and F-actin
2.8. Quantitative real-time PCR
For immunofluorescence microscopy of the α1 subunit of Na +/K +ATPase a separate group of rats was treated with venom for the times indicated above. At each interval, the rats were anesthetized and perfused with heparinized saline to remove residual blood from the kidneys which were then perfused with 4% paraformaldehyde in phosphate-buffered saline PBS, pH 7.4, in situ for 20 min. The kidneys were subsequently removed, sectioned sagitally and immersed in 4% paraformaldehyde for a further 2 h, and then washed sequentially with phosphate-buffered saline (PBS) (10 min), PBS + 1% glycine (30 min) and PBS + 15% sucrose (overnight). The tissues were subsequently embedded in tissue freezing medium, frozen in liquid N2 and stored at −80 °C. Sections were cut on a cryostat and washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 15 min at room temperature. Nonspecific binding sites were blocked by incubating the sections with 8% BSA (in PBS, pH 7.4) for 30 min at room temperature followed by washing with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 15 min at room temperature. After washing, the sections were incubated overnight at 4 °C with mouse monoclonal antibody to the α1 subunit of Na +/K +-ATPase (diluted 1:400 in PBS containing 1% BSA). The sections were subsequently washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 30 min at room temperature. After a further wash, the sections were incubated with goat anti-mouse FITC-conjugated secondary antibody (diluted 1:75 in PBS) for 3 h at room temperature in the dark. Finally, the sections were washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 30 min, in the dark, and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) prior to examination. The sections were evaluated with a Leica DM 5000 B fluorescence microscope. Images were captured with a CCD camera and analyzed with Leica Q Win Plus v.3.2.0 software. F-actin was detected by staining for 2 h with TRITC-conjugated phalloidin (diluted 1:80 in PBS), as previously described [38]. The sections were observed using a confocal laser-scanning microscope (LSM 510 META, Zeiss, Hamburg, Germany).
Total RNA was isolated from kidneys using Trizol according to the manufacturer's recommendations. RNA concentrations were determined based on the absorbance at 260 nm and the purity was determined by calculating the absorbance ratio at 260 nm and 280 nm, and by ethidium bromide staining of 1% agarose gels. Isolated RNA was stored at − 70 °C until analyzed by using the real-time polymerase chain reaction (PCR). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). The primers for the α1 subunit of Na +/K+-ATPase (Sense: 5′-CAGTGTTTCAGGCTAACCAAGAAA-3′; Anti-sense: 5′-CGCCGACTCGGAAGCAT-3′) and for transforming growth factor β1 (TGF-β1) (Sense: 5′-CAGTGTTTCAGGCTAACCAAGAAA-3′; Anti-sense: 5′-CCCGAATGTCTGACGTATTGAA-3′) were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA) and were purchased from Applied Biosystems. Real time PCR was done using SYBR Green master kits (Applied Biosystems) and 5 ng of cDNA/μl of sample. The fluorescence was quantified and the amplification plots were analyzed with a 7500 Sequence Detection System (Applied Biosystems). Na+/ K +-ATPase and TGF-β1 gene expressions were quantified according to Heid et al. [36] and Vandesompele et al. [37], respectively. Two replicates were run on the same plate for each sample and each sample was run twice, independently. The internal housekeeping (control) genes used in these assays were β-actin and GAPDH (for Na+/K +ATPase) and 18S (for TGF-β1).
Control
A
B. alternatus venom (0.8 mg/kg, i.v)
Area of capsular space (µm2)
4500
*
4000
*
*
*
3500 3000
*
2500
2.10. Statistical analysis
2000 1500 1000 500 0
6
48
24
72
7 days
Hours
B
Area (µm2)
3. Results
15000 12500
* *
* * *
3.1. Histological alterations
10000 7500 5000 2500 0
The results were expressed as the mean ± S.D., where appropriate, and statistical comparisons were done by using one-way analysis of variance (ANOVA) for repeated measurements followed by the Bonferroni test. A value of p b 0.05 indicated significance. All data analyses were done with Prism v4.0 software (GraphPad Inc., La Jolla, CA, USA).
6 24 48 72 7days
Hours Bowman's capsule
6 24 48 72 7days
Hours Glomerular tuft
Fig. 2. The increase in renal capsular space caused by B. alternatus venom. Fifteen to 20 glomeruli from one section per rat were analyzed as described in Methods. The columns are the mean ± S.D. of six rats. *p b 0.05 vs. the control group (ANOVA followed by the Bonferroni test).
Light microscopy of kidneys from saline-injected rats revealed a normal renal parenchyma (Fig. 1A). In contrast, the venom produced marked changes in the renal corpuscles (Fig. 1B–F) when compared to control tissue (Fig. 1A). These changes included lobulation of the capillary tufts (Fig. 1B,C,E), dilation of Bowman's space (Fig. 1B,C,F) and the presence of microaneurysms (Fig. 1C,D,F), as well as nodules that formed a dense, strongly-stained mesangial matrix (Fig. 1B,E). The severity of these lesions varied, with some capillary tufts showing a diffuse mesangial matrix. There was a significant increase in the area of the capsular space after envenoming (Fig. 2A). This increase was related to the distension of Bowman's capsule rather than a decrease in glomerular diameter (Fig. 2B) and was observed at all time intervals examined. The proximal and distal tubules had swollen lumens with cellular debris and amorphic material; there was also dilation of the peritubular
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capillaries (Fig. 1D,F). Other degenerative changes, such as thickening of the brush border and the presence of a discontinuous epithelium (Fig. 3B), as well as cell detachment with a loss of cell cytoplasm in proximal tubules (Fig. 3D) were also seen after envenoming, when compared to the control group (Fig. 3A,C). Confocal microscopy of TRITC-phalloidin-labeled sections of renal cortical tissue from control rats showed a normal cytoskeleton (Fig. 4A,E). In contrast, treatment with B. alternatus venom resulted in the disruption of actin filaments in the brush border (Fig. 4B,D), with diffuse labeling of the renal tubules (Fig. 4C,G). Bowman's capsule also showed disruption of the cytoskeleton, an increase in the capsular space and microaneurysms (Fig. 4F–H). Polarized light microscopy of sections stained with Sirius red revealed extensive pericorpuscular collagen deposition 24 h, 48 h and 72 h and 7 days after administration of B. alternatus venom (Fig. 5C–F) compared to saline-treated (control) rats (Fig. 5A) and 6 h postvenom (Fig. 5B). Foci of intertubular collagen deposition were also observed in the renal cortex 24 h to 7 days post-venom (Fig. 6).
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also significant proteinuria in venom-treated rats at 6 h (control: 0.74 ± 0.17 mg total protein vs venom: 1.24 ± 0.34 mg; p b 0.05; n = 6 each) and 24 h (control: 0.63 ± 0.24 mg vs venom: 1.16 ± 0.25 mg; p b 0.05; n = 6 each) that returned to normal from 48 h onwards. In addition, the fractional urinary sodium excretion (FENa +) was significantly higher at 6 h and 7 days in venom-treated rats compared to the control group (Fig. 7B). The increase in FENa + in venom-treated rats was accompanied by a significant increase in fractional urinary potassium excretion (FEK +) at 6 h (Fig. 7C). Likewise, the transient increase in urinary sodium excretion in venom-treated rats was accompanied by increased lithium clearance 6 h post-venom (Fig. 7D). This transiently enhanced lithium clearance was followed by a significant increase in fractional proximal sodium excretion (FEPNa+) at 6 h (Fig. 7E). There was also an increase in the fractional post-proximal sodium excretion (FEPPNa +) 72 h and 7 days after venom administration (Fig. 7F), but this increase was not significant. At none of the time intervals studied was there any significant change in the blood pressure of venom-treated rats (Table 1).
3.2. TGF-β1 expression 3.4. Renal Na +/K +-ATPase expression and activity in venom-treated rats Real-time PCR revealed enhanced TGF-β1 gene expression (arbitrary units; mean ± SD; n = 3–4) at 6 h (0.58 ± 0.27), 24 h (0.54 ± 0.11) and 48 h (0.70 ± 0.31) post-venom compared to control tissue (0.20 ± 0.15); gene expression at 72 h (0.36 ± 0.19) and 7 days postvenom (0.18 ± 0.07) was similar to control tissue. 3.3. Renal function There was a significant decrease in the glomerular filtration rate (estimated from the creatinine clearance) 24–48 h after venom administration when compared to control rats (Fig. 7A). There was
Treatment with B. alternatus venom significantly increased the gene expression of the α1 subunit of Na +/K +-ATPase 6 h after envenoming, although by 24 h after venom administration the expression of this subunit had returned to basal levels (Fig. 8A). This enhanced gene expression was accompanied by a significant increase in renal Na +/K +-ATPase activity 6 h and 24 h after venom injection, compared to control rats (Fig. 8B). Immunofluorescence microscopy of control tissue revealed intense staining for the α1 subunit of Na +/K +-ATPase in the basal cell membrane of the distal tubule and weak staining in proximal tubules and collecting
A
B
C
D
Fig. 3. Renal cortical sections stained with periodic acid-Schiff (A,B) and Sirius red (C,D). The sections are from rats treated with saline (A,C) and 6 h (B) and 7 days (D) after the injection of B. alternatus venom. Note the normal brush border in the proximal tubule (p) in (A) and discontinuous epithelium of the brush border (dashed arrows) in (B). There was cell detachment and cellular debris (solid arrows) in the proximal tubule (p) after treatment with venom in (D). Bars: 10 μm in (A) (same scale for B) and 30 μm in (C) (same scale for D).
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A
B
C
D
E
F
G
H
Fig. 4. Renal cortical sections stained with TRITC-conjugated phalloidin. The sections were obtained from saline-treated rats (A,E) and 6 h (B,F), 24 h (C,G) and 7 days (D,H) after the injection of B. alternatus venom. Note the normal brush border in the proximal tubule in (A), and the thinning and loss of the brush border (large arrows in B and D) and rupture of actin filaments (small arrows in C, F and G) after treatment with venom. Note also the increase in the glomerular capsular space (cs) in (G) and microaneurysms (m) in (F) and (H). Panels (B) and (F) show proximal tubules with a reduced lumen (*). Bars: 20 μm (for all panels).
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A
B
C
D
E
F
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Fig. 5. Polarized light micrographs showing pericorpuscular collagen fiber deposition in the renal cortex of rats 6 h (B), 24 h (C), 48 h (D) and 72 h (E) and 7 days (F) after the injection of B. alternatus venom. There was very little collagen deposition in saline-treated rats (A) and 6 h after venom (B). Bar: 30 μm (for all panels).
ducts (data not shown). Immunostaining for the α1 subunit was markedly enhanced 6 h after venom administration (Fig. 9C,D) when compared to saline-treated controls (Fig. 9A,B). At 24 h post-venom (Fig. 9E,F), the expression was still increased relative to the control, but was weaker than at 6 h.
A
4. Discussion The treatment of rats with B. alternatus venom produced extensive alterations in renal morphology, including brush border disorganization, tubular swelling, dilation of the peritubular capillaries and cell
B
Fig. 6. Polarized light micrographs showing foci of intertubular collagen deposition in the renal cortex of rats 24 h (A) and 7 days (B) after the injection of B. alternatus venom. Bar: 30 μm (both panels).
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Control B. alternatus venom (0.8 mg/kg, i.v)
Creatinine clearance
A
B
1250
0.25
1000
FENa+ (%)
0.20
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*
*
250
* *
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24
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72
7 days
6
Hours
D
12.5
*
FEK+ (%)
10.0 7.5
7 days
5.0
72
7 days
72
7 days
*
30 20 10 0
0.0 6
24
48
72
7 days
6
Hours
24
48
Hours
F
5
8
* FEPPNa+ (%)
4
FEPNa+ (%)
72
50 40
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E
48
Hours
Li Clearance
C
24
3 2 1
6 4 2 0
0 6
24
48
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7 days
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48
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Fig. 7. Changes in renal creatinine clearance (A), fractional sodium excretion (FENa ) (B), fractional potassium excretion (FEK+) (C), lithium clearance (D), fractional proximal sodium excretion (FEPNa+) (E) and fractional post-proximal sodium excretion (FEPPNa+) (F) caused by B. alternatus venom. Each point is the mean ± SD of six rats. *p b 0.05 vs. the corresponding control (ANOVA followed by the Bonferroni test).
death. These findings generally agreed with other animal studies showing that Bothrops venoms can damage renal glomeruli, proximal and distal tubules and basement membrane, with proliferation of the mesangial matrix, glomerular congestion and fibrin deposition [12– 15,39,40]. In the renal corpuscle, there was lobulation of the glomerular capillary tufts, formation of microaneurysms and an increase in
Table 1 Systolic arterial blood pressure in rats treated with saline or B. alternatus venom (0.8 mg/kg, i.v.). There were no significant changes in blood pressure in the venomtreated rats compared to control rats. Blood pressure (mm Hg)
Saline (control) Venom (0.8 mg/kg, i.v.)
6h
24 h
48 h
72 h
7d
136 ± 23 123 ± 11
128 ± 19 128 ± 9
126 ± 12 125 ± 14
119 ± 10 127 ± 10
132 ± 8 131 ± 8
The values are the mean ± SD of six rats per interval for each treatment (saline and venom).
capsular diameter, but no change in glomerular diameter. Renal tubules and Bowman's capsule also showed disruption of the cytoskeleton. The discontinuity of actin filaments in Bowman's capsule and other regions of the renal corpuscle could explain the increase in capsular diameter, the disappearance of filtration slits and impairment of the filtration barrier. Boer-Lima et al. [15] observed that after treatment of rats with B. moojeni venom the glomerular basement membrane was less homogenous, with discontinuities and thinning. In the same study, these authors also observed capillary ballooning and the formation of microaneurysms. Although the mechanisms involved in the formation of glomerular microaneurysms are poorly understood, a failure in the supporting system (mesangium, podocytes and glomerular basement membrane) of the glomerular tuft after damage induced by venom metalloproteinases and PLA2 could be a cause. The changes in renal function indicated that B. alternatus venom caused tubular and glomerular alterations compatible with acute renal failure. There was a decrease in creatinine clearance (an indicator of altered glomerular filtration rate), proteinuria, and an
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Control
A
B. alternatus venom (0.8 mg/kg, i.v)
(arbitrary units)
Gene expression
2.5
*
2.0 1.5 1.0 0.5 0.0 6
24
48
72
7 days
72
7 days
Hours
(nmol/mg/min)
Na+/K+ -ATPase activity
B
400
*
*
300
200
100
0 6
24
48
Hours Fig. 8. Gene expression of the α1 subunit of Na+/K+-ATPase (A) and Na+/K+-ATPase activity (B) in renal tissue of rats injected with B. alternatus venom. Gene expression was evaluated by real time PCR and the results were expressed in arbitrary units. Enzymatic activity was assayed as described in Section 2.7 and expressed as nmol of Pi released/mg of protein/min. The columns are the mean ± S.D. (n = 6). *p b 0.05 vs. the control group (ANOVA followed by the Bonferroni test).
increase in tubular sodium rejection. Since there were no changes in blood pressure at the various intervals studied, hypoperfusion and the resulting ischemia were unlikely to be a major factor in the development of this acute renal failure. However, since blood pressure measurements were not obtained prior to 6 h, we cannot exclude the possibility that the morphological and functional changes observed here were not influenced by renal ischemia following venom-induced hypotension at an earlier stage. Polarized light microscopy of Sirius red-stained sections revealed extensive collagen deposition in cortical periglomerular and peritubular regions within 24 h after venom administration that persisted up to seven days post-venom. Enhanced deposition of extracellular matrix (ECM) proteins in renal tissue has been observed in response to a variety of stimuli, including transforming growth factor β (TGF-β), cytokines (tumor necrosis factor (TNF)-α and -β, interleukin (IL)-1), several adhesion molecules and chemoattractants. These stimuli can also increase the levels of tissue inhibitors of matrix metalloproteinases, thereby attenuating ECM turnover [41–43]. In view of experimental evidence that Bothrops venoms [44–46] and purified components, e.g., myotoxic PLA2 and metalloproteinases [47– 49], can enhance local and systemic concentrations of pro-inflammatory factors such as TNF-α, IL-1β, IL-6 and IL-10, it is possible that these mediators could contribute to the renal collagen deposition seen in rats treated with B. alternatus venom. In the case of TGF-β1, the formation of this mediator can occur as a direct consequence of a loss of ECM integrity (reviewed in [50]), such as caused by Bothrops venoms and their metalloproteinases [45,51,52]. TGF-β1 can up-regulate the synthesis of ECM components and enhance the restoration of epithelial coverage in regenerating
903
tubules; this mediator also regulates epithelial tubular cell proliferation and differentiation [50]. Together, these effects could contribute to the enhanced collagen deposition seen here following the administration of B. alternatus venom. There was an increase in proximal sodium excretion 6 h after venom, with no changes in the fractional post-proximal sodium excretion at the same interval. Hence, the natriuretic response seen 6 h after venom probably resulted from functional impairment in the proximal tubule and was most likely related to the brush border disorganization noted here. The histological alterations seen in postproximal nephron segments suggested compensatory sodium reabsorption, which agreed with the striking enlargement of the distal tubules. This dilation probably occurred in response to a rise in distal sodium and water delivery provoked by rejection of this ion by the proximal tubule. In addition, an increase in potassium excretion also indicated increased sodium reabsorption in the post-proximal nephron since sodium reabsorption in the distal segment of renal tubules induces potassium excretion. Beyond 6 h post-venom, sodium reabsorption in proximal nephron segments (FEPNa +) returned to baseline values. There was a sustained increase in the fractional sodium excretion (FENa +) at seven days after envenoming. This increase in FENa + was followed by an elevated post-proximal sodium rejection. Together, these findings suggest that there may be a delayed or late phase nephrotoxicity in this renal tubule segment. The changes in brush border continuity and organization and in sodium handling by proximal tubular cells described above may result initially from cytoskeletal alterations. B. alternatus venom caused F-actin disruption in Bowman's capsule and in the brush border of renal tubules. In animal models of acute renal failure, dramatic alterations in the cytoskeleton of tubular epithelial cells occur rapidly and impair cell–cell and cell–matrix adhesion [53]. In renal endothelial cells, F-actin degradation can cause the loss of polarity in the expression of integrins, with consequent cell detachment from the underlying basement membrane [54]. Our findings agree with studies showing that the treatment of MDCK cells with B. moojeni [17] and B. alternatus [18] venoms resulted in the disruption of F-actin and subsequent cell detachment. The actin cytoskeleton is critical for maintaining cell structure and function, including microvilli in the proximal epithelium, cell polarity and the proper localization and function of vital proteins such as Na +/K +-ATPase, tight junctional proteins and cell–cell adhesion molecules [17,55,56]. Treatment with B. alternatus venom significantly enhanced tubular gene expression and protein content of the α1 subunit (the catalytic subunit responsible for sodium and potassium transport) of renal Na +/K +-ATPase, associated with increased enzyme activity within 24 h after envenoming. This finding agrees with Schaffazick et al. [57] who observed that the injection of Bothrops jararacussu venom in mouse extensor digitorum longus muscle produced a rapid increase in expression of the α1 subunit of Na +/K +-ATPase. The mechanisms involved in the venom-induced enhancement of renal Na +/K +ATPase expression and activity are unknown but could involve the same venom components responsible for the venom-induced morphological and functional alterations. In particular, altered membrane permeability to calcium through the action of venom PLA2 [58] could lead to cytoskeletal alterations and changes in the expression and activity of nephron Na +/K +-ATPase in an attempt to indirectly counteract the Ca 2+ overload, i.e., Ca 2+ extrusion through the Na +/ Ca 2+ exchanger operating in forward mode. The cytoskeletal disruption and enlargement of the distal tubules seen in vivo was accompanied by an increase in Na +/K +-ATPase expression and activity, possibly as part of a mechanism by the renal cortex to compensate for renal damage. The natriuretic response observed after treatment with B. alternatus venom could enhance Na +/K +-ATPase expression and activity in post-proximal segments of intact nephron. This increased pump activity could in turn lead to greater sodium reabsorption in the post-proximal segments.
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A
B
C
D
E
F
Fig. 9. Expression of the α1 subunit of Na+/K+-ATPase assessed by immunofluorescence in renal cortex in saline-treated rats (controls) (A,B) and 6 h (C,D) and 24 h (E,F) after the injection of B. alternatus venom. Note the intense staining for the α1 subunit of Na+/K+-ATPase after 6 h (C,D) compared to control tissue (A,B). Twenty-four hours after venom (E,F), the staining was less intense than after 6 h, but was still greater than in control tissue (A,B). Panels (C) and (E) are the same magnification as (A), and (D) and (F) are the same magnification as (B). Bars: 30 μm.
Based on these considerations, we suggest that the enhanced Na +/K +-ATPase expression and activity seen after the injection of B. alternatus venom may be a protective mechanism aimed at preserving renal function during acute renal damage. This conclusion agrees with Tomaz et al. [59] who found that inhibition of Na+–K +-ATPase activity with ouabain potentiated the myonecrosis (measured as creatine kinase release) caused by B. jararacussu venom in mouse skeletal muscle in vitro, i.e., normal functioning of the enzyme attenuated myonecrosis. As noted above, rats injected with B. alternatus venom showed signs of renal failure such as proteinuria and reduced creatinine clearance 6–48 h post-venom. The time scale of these changes in renal function agrees with the onset of renal dysfunction seen in clinical studies of Bothrops bites in which renal failure occurs 18 h to 5 days after envenoming (within 24 h in 56% of patients) [8,9]. Although the high number of variables associated with envenoming in humans (patient and snake age, amount of venom injected, site of bite, first aid measures used, time to hospital admission, use of antivenom) makes it difficult to directly compare clinical cases with controlled experimental studies, it is nevertheless interesting to note that the alterations in Na +/K +-ATPase expression and activity seen here
(within 24 h) coincided with the changes in renal function seen experimentally and clinically. In conclusion, B. alternatus venom causes significant morphological and functional changes in rat renal tissue, with enhanced Na+/K+-ATPase expression and activity. This increase in Na+/K +-ATPase expression and activity may attenuate renal dysfunction during venom-induced damage.
Role of funding source None of the funding agencies indicated in the acknowledgements was involved in the study design, data collection, analysis and interpretation, or in the preparation and writing of the article and the decision to submit the paper for publication.
Conflict of interest statement None of the authors have any conflicts of interest related to this work.
A. Linardi et al. / Biochimica et Biophysica Acta 1810 (2011) 895–906
Acknowledgements The authors thank José Ilton dos Santos, Maiara Daguana and Marta Beatriz Leonardo for technical assistance, and Dr. Maria Alice da Cruz-Höfling (Departamento de Histologia e Embriologia, IB, UNICAMP) for providing laboratory space and advice in the early stages of this work. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). K.C.C. and T.A.A.R.S. were supported by doctoral studentships and A.L. and E.H.M. were supported by post-doctoral fellowships from FAPESP. S.H. is supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] F.O.S. França, C.M.S. Málaque, Acidente botrópico, in: J.L.C. Cardoso, F.O.S. França, F.H. Wen, C.M.S. Málaque, V. Haddad Jr. (Eds.), Animais Peçonhentos no Brasil: Biologia, Clínica e Terapêutica dos Acidentes, Sarvier/FAPESP, São Paulo, 2003, pp. 72–86. [2] D.A. 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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na +/K +-ATPase Alessandra Linardi a, b, Thomaz A.A. Rocha e Silva a, b, Elen H. Miyabara c, Carla F. Franco-Penteado d, Kiara C. Cardoso a, Patrícia A. Boer e, Anselmo S. Moriscot f, José A.R. Gontijo e, Paulo P. Joazeiro g, Carla B. Collares-Buzato g, Stephen Hyslop a,⁎ a
Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil Departamento de Ciências Fisiológicas, Faculdade de Ciências Médicas da Santa Casa de São Paulo, 01221-020, São Paulo, SP, Brazil c Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), 05508-900, São Paulo, SP, Brazil d Hemocentro, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil e Núcleo de Medicina e Cirurgia Experimental, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil f Departamento de Biologia Celular e Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo (USP), 05508-900, São Paulo, SP, Brazil g Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13083-970, Campinas, SP, Brazil b
a r t i c l e
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Article history: Received 14 February 2011 Received in revised form 23 May 2011 Accepted 1 June 2011 Available online 24 June 2011 Keywords: Acute renal failure Bothrops alternatus Collagen deposition F-actin Na+/K+-ATPase Renal function
a b s t r a c t Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na +/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na +/K +-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na +/K +-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na +/K +-ATPase α1 subunit were increased 6 h post-venom, whereas Na +/K+-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na +/K +-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na +/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Envenoming by Bothrops snakes can cause acute renal failure [1–3], with an overall incidence ranging from b3% [4–6] to ~ 38% [7]. Acute renal failure is an important cause of death among patients bitten by Bothrops species (accounting for ~ 80% of deaths in some series) [7–10]. Clinically, venom-induced acute renal failure may be caused by hypovolemia or hypoperfusion of renal tissue, a direct nephrotoxic action of venom components, or disseminated intravas-
⁎ Corresponding author. Tel.: + 55 19 3521 9536; fax: + 55 19 3289 2968. E-mail address: [email protected] (S. Hyslop). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.006
cular coagulation. The resulting histological renal damage involves acute glomerulonephritis or acute tubular or cortical necrosis, while the functional alterations include metabolic acidosis, hyperkalemia, hematuria, reduced urine output (oliguria or anuria) and elevated serum urea and creatinine concentrations [3,7–9,11]. Experimentally, Bothrops venoms can damage renal glomeruli, proximal and distal tubules and the basement membrane, with proliferation of the mesangial matrix, glomerular congestion, brush border disorganization and fibrin deposition [12–16]. Studies using cultured Madin-Darby canine kidney (MDCK) cells (a renal epithelial cell line) have shown that Bothrops venoms are directly cytotoxic to renal cells by mechanisms that involve extensive disruption of the cytoskeleton and cell death by necrosis [17,18].
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In addition to morphological damage, Bothrops venoms also cause functional alterations. In rats, Bothrops jararaca venom causes acute renal failure, with a reduction in the glomerular filtration rate, osmotic clearance and renal plasma flow, and an increase in serum creatinine levels; there are variable effects on sodium excretion and mean arterial blood pressure [12,13]. Similar studies with Bothrops moojeni venom have also described glomerular damage, tubular degeneration and desquamation, hematuria and a reduction in glomerular filtration rate, with increments in urinary sodium excretion, but no changes in arterial blood pressure and no fibrin deposition in glomerular capillaries [14,15]. These studies indicate that acute renal failure caused by Bothrops venoms is characterized by impaired renal function that involves a reduction in glomerular filtration and tubular sodium reabsorption. Renal Na +/K +-ATPase plays a pivotal role in the active transport of certain solutes and maintenance of intracellular electrolyte homeostasis, with functional alterations in this pump leading to a reduction in glomerular filtration and tubular sodium reabsorption [19]. The localization of Na +/K +-ATPase in the basolateral membrane is regulated by direct interactions with membrane-associated cytoskeletal proteins, and this polar distribution of Na +/K +-ATPase is essential for efficient enzyme function and sodium reabsorption by renal tubular cells [20,21]. Since the accumulation of Bothrops venom in the kidney [22,23] may contribute to acute renal failure experimentally and clinically, and since renal Na +/K +-ATPase is important for maintaining intracellular electrolyte homeostasis, such as sodium reabsorption by renal tubular cells [20,21], in this study we investigated the morphological and functional renal alterations caused by B. alternatus venom in rats and the changes in renal Na +/K +-ATPase expression and activity. This is the first study to examine changes in renal Na +/K +-ATPase following the administration of Bothrops venom in vivo and to correlate these with venom-induced functional alterations. 2. Materials and methods 2.1. Reagents and venom Adenosine 5′-triphosphate (sodium salt), bovine serum albumin, goat anti-mouse FITC-conjugated secondary antibody, ouabain, paraformaldehyde and TRITC-conjugated phalloidin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse monoclonal antibody to the α1-subunit of Na +/K +-ATPase was purchased from Upstate (Lake Placid, NY, USA), glycine was from Amersham-GE Lifesciences (Piscataway, NJ, USA) and tissue freezing medium was from Leica (Nussloch, Germany). Commercial kits for creatinine quantification were from CELM (Barueri, SP, Brazil). Isoflurane was from Cristália (Itapira, SP, Brazil). The other reagents of analytical grade were obtained from the suppliers indicated in the appropriate sections below. Ninety-six well plates were from Corning (Corning, MA, USA). Bothrops alternatus venom obtained from adult snakes of both sexes was purchased from the Centro de Extração de Toxinas Animais (CETA, Morungaba, SP, Brazil) and was stored lyophilized at −20 °C. 2.2. Animals Male Wistar-Hanover rats (200–250 g) were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at UNICAMP and were housed 6 animals/cage at 23 °C on a 12 h light/ dark cycle, with free access to food and water. The experimental protocols were approved by an institutional Committee for Ethics in Animal Experimentation (CEEA/UNICAMP, protocol no. 681–1) and were done according to the general ethical guidelines for animal use
established by the Brazilian Society of Laboratory Animal Science (SBCAL) and EC Directive 86/609/EEC for Animal Experiments. 2.3. Venom dose and administration protocol A single venom dose of 0.8 mg/kg was used in this work based on preliminary experiments in which doses of 0.4 and 1.6 mg/kg were also tested. Rats injected with 0.4 mg/kg showed minimal histological alterations, whereas those receiving 1.6 mg/kg generally died within a few minutes or hours after venom administration — this short survival time precluded adequate assessment of changes in renal function. The dose of 0.8 mg/kg provided the best combination of renal damage (assessed histologically) in relation to survival time. This dose was similar to that used in other experimental studies, e.g., 0.4 mg/kg, i.v., for Bothrops jararaca [13] and Bothrops moojeni [14,15] and broadly agreed with the clinical setting. Since B. alternatus produces venom yields of 60–400 mg [24–27], the injection of all available venom in a bite would correspond to a theoretical dose of 0.86–5.71 mg/kg in a 70 kg human, which is probably greater than the true range of doses. With the injection of only 50% of the available venom, the dose in humans would be 0.43–2.85 mg/kg, a range that includes the dose of 0.8 mg/kg used here. Based on these considerations, the venom dose used here was not unrealistic and was probably very close to the clinical situation. Rats (n = 6/group) were injected with venom (0.8 mg/kg, i.v.) or 0.15 M NaCl (control) and 6, 24, 48 and 72 h and 7 days later they were anesthetized with isoflurane and killed by exsanguination, after which the kidneys were removed and immediately immersed in the appropriate fixative for further tissue processing or frozen in liquid nitrogen and stored at − 80 °C. 2.4. Histological analysis At the intervals indicated above, the kidneys were removed and cut sagitally prior to fixation in buffered 10% formol solution for 24 h, followed by dehydration in a graded ethanol series and embedding in Histosec (Merck, Rio de Janeiro, RJ, Brazil). Sections 5 μm thick were stained with periodic acid-Schiff or Sirius red reagent for examination by light microscopy and polarized light microscopy, respectively. Computer-assisted morphometric analysis was done using a Nikon Eclipse E800 microscope and Image ProPlus software. Glomerular damage was quantified by measuring the area (μm 2) after manual tracing of each Bowman's capsule and the corresponding glomerular tuft in 15–20 randomly selected renal corpuscles in one section from each of six rats per group. The capsular space was calculated as the difference between the glomerular tuft area and Bowman's capsule area [28]. 2.5. Renal function Rats were injected with B. alternatus venom (0.8 mg/kg, i.v.) or 0.15 M NaCl (control) and renal function was studied at the intervals mentioned above using a LiCl (60 mmol/100 g of body weight) tap water load given by gavage (2% of body weight), as described elsewhere [14]. At the end of the experiment, the rats were anesthetized and blood samples (obtained by cardiac puncture) and urine samples were collected for analysis (see below). Arterial blood pressure was measured by a non-invasive tail-cuff method [29] immediately before the rats were killed. Plasma and urinary sodium and potassium concentrations were measured by flame photometry (model B262; Micronal, São Paulo, SP, Brazil). Creatinine was determined by the alkaline picrate method [30] using a SpectraMax340 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA) and proteinuria was determined according to Bradford [31], with bovine serum albumin as the standard. Renal clearance (C) was calculated by a standard formula
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(C = UV/P) using the plasma creatinine and lithium levels for each period, where U = urinary concentration, V = urinary volume and P = plasma concentration. Creatinine clearance was used to estimate the glomerular filtration rate and lithium clearance (CLi +) was used to assess sodium proximal tubule output. Fractional sodium (FENa +) and potassium (FEK +) excretions were calculated as CNa +/CCr and CK +/CCr, respectively, where CNa + and CK + are the ion clearances and CCr is the creatinine clearance. Fractional proximal (FEPNa +) and post-proximal (FEPPNa +) sodium excretions were calculated as CLi +/ CCr × 100 and CNa +/CLi + × 100, respectively [14].
and processed through two more cycles of homogenization and centrifugation. The final pellet was discarded and the pooled supernatants were centrifuged at 10,000 ×g for 15 min. The new supernatant was discarded and the pellet was resuspended in 2 ml of the same homogenization solution, aliquoted and stored at −80 °C for enzyme activity. Protein concentrations were determined according to Bradford [31].
2.6. Tissue processing
Na +/K +-ATPase activity was assayed as described by Forbush [32]. For this assay, 100 μl of sample was added to 500 μl of buffer containing 60 mM Tris–HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 4 mM Na2ATP, 4 mM MgCl2 and 1 mM EDTA followed by incubation for 10 min at 37 °C. Na +/K +-ATPase activity was calculated as the difference between the inorganic phosphate (Pi) released in control tubes and that released in identical samples containing 1 mM ouabain. Inorganic phosphate was determined according to Baginski et al. [33], as modified by Ottolenghi [34] and Brotherus et al. [35]. The resulting
One kidney from each pair of kidneys stored at − 80 °C was used to assay Na +/K +-ATPase activity (Section 2.7) while the second kidney was used for RNA isolation and real time PCR (Section 2.8). For Na +/ K +-ATPase activity, the kidney was homogenized in 0.05 M Tris–HCl, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA, centrifuged (1000 ×g, 10 min, 4 °C), and the supernatant was collected and stored on ice. The pellet was resuspended in half the original volume
A
2.7. Na +/K +-ATPase assay
B
p
* d
D
C
d v
* p
m m md
E
p F
* m
d p
Fig. 1. Renal cortical sections stained with Sirius red. The sections were obtained from saline-treated (control) rats (A) and 6 h (B), 24 h (C), 48 h (D), 72 h (E) and 7 days (F) after the injection of B. alternatus venom. Note the hypertrophied renal corpuscles and nodules that form a densely clumped, strongly stained mesangial matrix (solid arrows) and widening of the capsular space (*) in venom-treated rats. Note also the distended distal (d) and proximal (p) tubules containing cellular debris and amorphic material, and the dilated peritubular capillaries (dashed arrows). m - microaneurysms, md - macula densa, v - vascular pole. Bar: 30 μm (for all panels).
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absorbance was measured at 705 nm and the activity was expressed as nmol of Pi released/mg of protein/min.
2.9. Fluorescence microscopy of the α1 subunit of Na +/K +-ATPase and F-actin
2.8. Quantitative real-time PCR
For immunofluorescence microscopy of the α1 subunit of Na +/K +ATPase a separate group of rats was treated with venom for the times indicated above. At each interval, the rats were anesthetized and perfused with heparinized saline to remove residual blood from the kidneys which were then perfused with 4% paraformaldehyde in phosphate-buffered saline PBS, pH 7.4, in situ for 20 min. The kidneys were subsequently removed, sectioned sagitally and immersed in 4% paraformaldehyde for a further 2 h, and then washed sequentially with phosphate-buffered saline (PBS) (10 min), PBS + 1% glycine (30 min) and PBS + 15% sucrose (overnight). The tissues were subsequently embedded in tissue freezing medium, frozen in liquid N2 and stored at −80 °C. Sections were cut on a cryostat and washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 15 min at room temperature. Nonspecific binding sites were blocked by incubating the sections with 8% BSA (in PBS, pH 7.4) for 30 min at room temperature followed by washing with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 15 min at room temperature. After washing, the sections were incubated overnight at 4 °C with mouse monoclonal antibody to the α1 subunit of Na +/K +-ATPase (diluted 1:400 in PBS containing 1% BSA). The sections were subsequently washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 30 min at room temperature. After a further wash, the sections were incubated with goat anti-mouse FITC-conjugated secondary antibody (diluted 1:75 in PBS) for 3 h at room temperature in the dark. Finally, the sections were washed with PBS containing 0.3% Triton-X 100 and 0.1% BSA for 30 min, in the dark, and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) prior to examination. The sections were evaluated with a Leica DM 5000 B fluorescence microscope. Images were captured with a CCD camera and analyzed with Leica Q Win Plus v.3.2.0 software. F-actin was detected by staining for 2 h with TRITC-conjugated phalloidin (diluted 1:80 in PBS), as previously described [38]. The sections were observed using a confocal laser-scanning microscope (LSM 510 META, Zeiss, Hamburg, Germany).
Total RNA was isolated from kidneys using Trizol according to the manufacturer's recommendations. RNA concentrations were determined based on the absorbance at 260 nm and the purity was determined by calculating the absorbance ratio at 260 nm and 280 nm, and by ethidium bromide staining of 1% agarose gels. Isolated RNA was stored at − 70 °C until analyzed by using the real-time polymerase chain reaction (PCR). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). The primers for the α1 subunit of Na +/K+-ATPase (Sense: 5′-CAGTGTTTCAGGCTAACCAAGAAA-3′; Anti-sense: 5′-CGCCGACTCGGAAGCAT-3′) and for transforming growth factor β1 (TGF-β1) (Sense: 5′-CAGTGTTTCAGGCTAACCAAGAAA-3′; Anti-sense: 5′-CCCGAATGTCTGACGTATTGAA-3′) were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA) and were purchased from Applied Biosystems. Real time PCR was done using SYBR Green master kits (Applied Biosystems) and 5 ng of cDNA/μl of sample. The fluorescence was quantified and the amplification plots were analyzed with a 7500 Sequence Detection System (Applied Biosystems). Na+/ K +-ATPase and TGF-β1 gene expressions were quantified according to Heid et al. [36] and Vandesompele et al. [37], respectively. Two replicates were run on the same plate for each sample and each sample was run twice, independently. The internal housekeeping (control) genes used in these assays were β-actin and GAPDH (for Na+/K +ATPase) and 18S (for TGF-β1).
Control
A
B. alternatus venom (0.8 mg/kg, i.v)
Area of capsular space (µm2)
4500
*
4000
*
*
*
3500 3000
*
2500
2.10. Statistical analysis
2000 1500 1000 500 0
6
48
24
72
7 days
Hours
B
Area (µm2)
3. Results
15000 12500
* *
* * *
3.1. Histological alterations
10000 7500 5000 2500 0
The results were expressed as the mean ± S.D., where appropriate, and statistical comparisons were done by using one-way analysis of variance (ANOVA) for repeated measurements followed by the Bonferroni test. A value of p b 0.05 indicated significance. All data analyses were done with Prism v4.0 software (GraphPad Inc., La Jolla, CA, USA).
6 24 48 72 7days
Hours Bowman's capsule
6 24 48 72 7days
Hours Glomerular tuft
Fig. 2. The increase in renal capsular space caused by B. alternatus venom. Fifteen to 20 glomeruli from one section per rat were analyzed as described in Methods. The columns are the mean ± S.D. of six rats. *p b 0.05 vs. the control group (ANOVA followed by the Bonferroni test).
Light microscopy of kidneys from saline-injected rats revealed a normal renal parenchyma (Fig. 1A). In contrast, the venom produced marked changes in the renal corpuscles (Fig. 1B–F) when compared to control tissue (Fig. 1A). These changes included lobulation of the capillary tufts (Fig. 1B,C,E), dilation of Bowman's space (Fig. 1B,C,F) and the presence of microaneurysms (Fig. 1C,D,F), as well as nodules that formed a dense, strongly-stained mesangial matrix (Fig. 1B,E). The severity of these lesions varied, with some capillary tufts showing a diffuse mesangial matrix. There was a significant increase in the area of the capsular space after envenoming (Fig. 2A). This increase was related to the distension of Bowman's capsule rather than a decrease in glomerular diameter (Fig. 2B) and was observed at all time intervals examined. The proximal and distal tubules had swollen lumens with cellular debris and amorphic material; there was also dilation of the peritubular
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capillaries (Fig. 1D,F). Other degenerative changes, such as thickening of the brush border and the presence of a discontinuous epithelium (Fig. 3B), as well as cell detachment with a loss of cell cytoplasm in proximal tubules (Fig. 3D) were also seen after envenoming, when compared to the control group (Fig. 3A,C). Confocal microscopy of TRITC-phalloidin-labeled sections of renal cortical tissue from control rats showed a normal cytoskeleton (Fig. 4A,E). In contrast, treatment with B. alternatus venom resulted in the disruption of actin filaments in the brush border (Fig. 4B,D), with diffuse labeling of the renal tubules (Fig. 4C,G). Bowman's capsule also showed disruption of the cytoskeleton, an increase in the capsular space and microaneurysms (Fig. 4F–H). Polarized light microscopy of sections stained with Sirius red revealed extensive pericorpuscular collagen deposition 24 h, 48 h and 72 h and 7 days after administration of B. alternatus venom (Fig. 5C–F) compared to saline-treated (control) rats (Fig. 5A) and 6 h postvenom (Fig. 5B). Foci of intertubular collagen deposition were also observed in the renal cortex 24 h to 7 days post-venom (Fig. 6).
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also significant proteinuria in venom-treated rats at 6 h (control: 0.74 ± 0.17 mg total protein vs venom: 1.24 ± 0.34 mg; p b 0.05; n = 6 each) and 24 h (control: 0.63 ± 0.24 mg vs venom: 1.16 ± 0.25 mg; p b 0.05; n = 6 each) that returned to normal from 48 h onwards. In addition, the fractional urinary sodium excretion (FENa +) was significantly higher at 6 h and 7 days in venom-treated rats compared to the control group (Fig. 7B). The increase in FENa + in venom-treated rats was accompanied by a significant increase in fractional urinary potassium excretion (FEK +) at 6 h (Fig. 7C). Likewise, the transient increase in urinary sodium excretion in venom-treated rats was accompanied by increased lithium clearance 6 h post-venom (Fig. 7D). This transiently enhanced lithium clearance was followed by a significant increase in fractional proximal sodium excretion (FEPNa+) at 6 h (Fig. 7E). There was also an increase in the fractional post-proximal sodium excretion (FEPPNa +) 72 h and 7 days after venom administration (Fig. 7F), but this increase was not significant. At none of the time intervals studied was there any significant change in the blood pressure of venom-treated rats (Table 1).
3.2. TGF-β1 expression 3.4. Renal Na +/K +-ATPase expression and activity in venom-treated rats Real-time PCR revealed enhanced TGF-β1 gene expression (arbitrary units; mean ± SD; n = 3–4) at 6 h (0.58 ± 0.27), 24 h (0.54 ± 0.11) and 48 h (0.70 ± 0.31) post-venom compared to control tissue (0.20 ± 0.15); gene expression at 72 h (0.36 ± 0.19) and 7 days postvenom (0.18 ± 0.07) was similar to control tissue. 3.3. Renal function There was a significant decrease in the glomerular filtration rate (estimated from the creatinine clearance) 24–48 h after venom administration when compared to control rats (Fig. 7A). There was
Treatment with B. alternatus venom significantly increased the gene expression of the α1 subunit of Na +/K +-ATPase 6 h after envenoming, although by 24 h after venom administration the expression of this subunit had returned to basal levels (Fig. 8A). This enhanced gene expression was accompanied by a significant increase in renal Na +/K +-ATPase activity 6 h and 24 h after venom injection, compared to control rats (Fig. 8B). Immunofluorescence microscopy of control tissue revealed intense staining for the α1 subunit of Na +/K +-ATPase in the basal cell membrane of the distal tubule and weak staining in proximal tubules and collecting
A
B
C
D
Fig. 3. Renal cortical sections stained with periodic acid-Schiff (A,B) and Sirius red (C,D). The sections are from rats treated with saline (A,C) and 6 h (B) and 7 days (D) after the injection of B. alternatus venom. Note the normal brush border in the proximal tubule (p) in (A) and discontinuous epithelium of the brush border (dashed arrows) in (B). There was cell detachment and cellular debris (solid arrows) in the proximal tubule (p) after treatment with venom in (D). Bars: 10 μm in (A) (same scale for B) and 30 μm in (C) (same scale for D).
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A
B
C
D
E
F
G
H
Fig. 4. Renal cortical sections stained with TRITC-conjugated phalloidin. The sections were obtained from saline-treated rats (A,E) and 6 h (B,F), 24 h (C,G) and 7 days (D,H) after the injection of B. alternatus venom. Note the normal brush border in the proximal tubule in (A), and the thinning and loss of the brush border (large arrows in B and D) and rupture of actin filaments (small arrows in C, F and G) after treatment with venom. Note also the increase in the glomerular capsular space (cs) in (G) and microaneurysms (m) in (F) and (H). Panels (B) and (F) show proximal tubules with a reduced lumen (*). Bars: 20 μm (for all panels).
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A
B
C
D
E
F
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Fig. 5. Polarized light micrographs showing pericorpuscular collagen fiber deposition in the renal cortex of rats 6 h (B), 24 h (C), 48 h (D) and 72 h (E) and 7 days (F) after the injection of B. alternatus venom. There was very little collagen deposition in saline-treated rats (A) and 6 h after venom (B). Bar: 30 μm (for all panels).
ducts (data not shown). Immunostaining for the α1 subunit was markedly enhanced 6 h after venom administration (Fig. 9C,D) when compared to saline-treated controls (Fig. 9A,B). At 24 h post-venom (Fig. 9E,F), the expression was still increased relative to the control, but was weaker than at 6 h.
A
4. Discussion The treatment of rats with B. alternatus venom produced extensive alterations in renal morphology, including brush border disorganization, tubular swelling, dilation of the peritubular capillaries and cell
B
Fig. 6. Polarized light micrographs showing foci of intertubular collagen deposition in the renal cortex of rats 24 h (A) and 7 days (B) after the injection of B. alternatus venom. Bar: 30 μm (both panels).
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Control B. alternatus venom (0.8 mg/kg, i.v)
Creatinine clearance
A
B
1250
0.25
1000
FENa+ (%)
0.20
750 500
*
*
250
* *
0.15 0.10 0.05 0.00
0 6
24
48
72
7 days
6
Hours
D
12.5
*
FEK+ (%)
10.0 7.5
7 days
5.0
72
7 days
72
7 days
*
30 20 10 0
0.0 6
24
48
72
7 days
6
Hours
24
48
Hours
F
5
8
* FEPPNa+ (%)
4
FEPNa+ (%)
72
50 40
2.5
E
48
Hours
Li Clearance
C
24
3 2 1
6 4 2 0
0 6
24
48
72
7 days
Hours
6
24
48
Hours +
Fig. 7. Changes in renal creatinine clearance (A), fractional sodium excretion (FENa ) (B), fractional potassium excretion (FEK+) (C), lithium clearance (D), fractional proximal sodium excretion (FEPNa+) (E) and fractional post-proximal sodium excretion (FEPPNa+) (F) caused by B. alternatus venom. Each point is the mean ± SD of six rats. *p b 0.05 vs. the corresponding control (ANOVA followed by the Bonferroni test).
death. These findings generally agreed with other animal studies showing that Bothrops venoms can damage renal glomeruli, proximal and distal tubules and basement membrane, with proliferation of the mesangial matrix, glomerular congestion and fibrin deposition [12– 15,39,40]. In the renal corpuscle, there was lobulation of the glomerular capillary tufts, formation of microaneurysms and an increase in
Table 1 Systolic arterial blood pressure in rats treated with saline or B. alternatus venom (0.8 mg/kg, i.v.). There were no significant changes in blood pressure in the venomtreated rats compared to control rats. Blood pressure (mm Hg)
Saline (control) Venom (0.8 mg/kg, i.v.)
6h
24 h
48 h
72 h
7d
136 ± 23 123 ± 11
128 ± 19 128 ± 9
126 ± 12 125 ± 14
119 ± 10 127 ± 10
132 ± 8 131 ± 8
The values are the mean ± SD of six rats per interval for each treatment (saline and venom).
capsular diameter, but no change in glomerular diameter. Renal tubules and Bowman's capsule also showed disruption of the cytoskeleton. The discontinuity of actin filaments in Bowman's capsule and other regions of the renal corpuscle could explain the increase in capsular diameter, the disappearance of filtration slits and impairment of the filtration barrier. Boer-Lima et al. [15] observed that after treatment of rats with B. moojeni venom the glomerular basement membrane was less homogenous, with discontinuities and thinning. In the same study, these authors also observed capillary ballooning and the formation of microaneurysms. Although the mechanisms involved in the formation of glomerular microaneurysms are poorly understood, a failure in the supporting system (mesangium, podocytes and glomerular basement membrane) of the glomerular tuft after damage induced by venom metalloproteinases and PLA2 could be a cause. The changes in renal function indicated that B. alternatus venom caused tubular and glomerular alterations compatible with acute renal failure. There was a decrease in creatinine clearance (an indicator of altered glomerular filtration rate), proteinuria, and an
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Control
A
B. alternatus venom (0.8 mg/kg, i.v)
(arbitrary units)
Gene expression
2.5
*
2.0 1.5 1.0 0.5 0.0 6
24
48
72
7 days
72
7 days
Hours
(nmol/mg/min)
Na+/K+ -ATPase activity
B
400
*
*
300
200
100
0 6
24
48
Hours Fig. 8. Gene expression of the α1 subunit of Na+/K+-ATPase (A) and Na+/K+-ATPase activity (B) in renal tissue of rats injected with B. alternatus venom. Gene expression was evaluated by real time PCR and the results were expressed in arbitrary units. Enzymatic activity was assayed as described in Section 2.7 and expressed as nmol of Pi released/mg of protein/min. The columns are the mean ± S.D. (n = 6). *p b 0.05 vs. the control group (ANOVA followed by the Bonferroni test).
increase in tubular sodium rejection. Since there were no changes in blood pressure at the various intervals studied, hypoperfusion and the resulting ischemia were unlikely to be a major factor in the development of this acute renal failure. However, since blood pressure measurements were not obtained prior to 6 h, we cannot exclude the possibility that the morphological and functional changes observed here were not influenced by renal ischemia following venom-induced hypotension at an earlier stage. Polarized light microscopy of Sirius red-stained sections revealed extensive collagen deposition in cortical periglomerular and peritubular regions within 24 h after venom administration that persisted up to seven days post-venom. Enhanced deposition of extracellular matrix (ECM) proteins in renal tissue has been observed in response to a variety of stimuli, including transforming growth factor β (TGF-β), cytokines (tumor necrosis factor (TNF)-α and -β, interleukin (IL)-1), several adhesion molecules and chemoattractants. These stimuli can also increase the levels of tissue inhibitors of matrix metalloproteinases, thereby attenuating ECM turnover [41–43]. In view of experimental evidence that Bothrops venoms [44–46] and purified components, e.g., myotoxic PLA2 and metalloproteinases [47– 49], can enhance local and systemic concentrations of pro-inflammatory factors such as TNF-α, IL-1β, IL-6 and IL-10, it is possible that these mediators could contribute to the renal collagen deposition seen in rats treated with B. alternatus venom. In the case of TGF-β1, the formation of this mediator can occur as a direct consequence of a loss of ECM integrity (reviewed in [50]), such as caused by Bothrops venoms and their metalloproteinases [45,51,52]. TGF-β1 can up-regulate the synthesis of ECM components and enhance the restoration of epithelial coverage in regenerating
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tubules; this mediator also regulates epithelial tubular cell proliferation and differentiation [50]. Together, these effects could contribute to the enhanced collagen deposition seen here following the administration of B. alternatus venom. There was an increase in proximal sodium excretion 6 h after venom, with no changes in the fractional post-proximal sodium excretion at the same interval. Hence, the natriuretic response seen 6 h after venom probably resulted from functional impairment in the proximal tubule and was most likely related to the brush border disorganization noted here. The histological alterations seen in postproximal nephron segments suggested compensatory sodium reabsorption, which agreed with the striking enlargement of the distal tubules. This dilation probably occurred in response to a rise in distal sodium and water delivery provoked by rejection of this ion by the proximal tubule. In addition, an increase in potassium excretion also indicated increased sodium reabsorption in the post-proximal nephron since sodium reabsorption in the distal segment of renal tubules induces potassium excretion. Beyond 6 h post-venom, sodium reabsorption in proximal nephron segments (FEPNa +) returned to baseline values. There was a sustained increase in the fractional sodium excretion (FENa +) at seven days after envenoming. This increase in FENa + was followed by an elevated post-proximal sodium rejection. Together, these findings suggest that there may be a delayed or late phase nephrotoxicity in this renal tubule segment. The changes in brush border continuity and organization and in sodium handling by proximal tubular cells described above may result initially from cytoskeletal alterations. B. alternatus venom caused F-actin disruption in Bowman's capsule and in the brush border of renal tubules. In animal models of acute renal failure, dramatic alterations in the cytoskeleton of tubular epithelial cells occur rapidly and impair cell–cell and cell–matrix adhesion [53]. In renal endothelial cells, F-actin degradation can cause the loss of polarity in the expression of integrins, with consequent cell detachment from the underlying basement membrane [54]. Our findings agree with studies showing that the treatment of MDCK cells with B. moojeni [17] and B. alternatus [18] venoms resulted in the disruption of F-actin and subsequent cell detachment. The actin cytoskeleton is critical for maintaining cell structure and function, including microvilli in the proximal epithelium, cell polarity and the proper localization and function of vital proteins such as Na +/K +-ATPase, tight junctional proteins and cell–cell adhesion molecules [17,55,56]. Treatment with B. alternatus venom significantly enhanced tubular gene expression and protein content of the α1 subunit (the catalytic subunit responsible for sodium and potassium transport) of renal Na +/K +-ATPase, associated with increased enzyme activity within 24 h after envenoming. This finding agrees with Schaffazick et al. [57] who observed that the injection of Bothrops jararacussu venom in mouse extensor digitorum longus muscle produced a rapid increase in expression of the α1 subunit of Na +/K +-ATPase. The mechanisms involved in the venom-induced enhancement of renal Na +/K +ATPase expression and activity are unknown but could involve the same venom components responsible for the venom-induced morphological and functional alterations. In particular, altered membrane permeability to calcium through the action of venom PLA2 [58] could lead to cytoskeletal alterations and changes in the expression and activity of nephron Na +/K +-ATPase in an attempt to indirectly counteract the Ca 2+ overload, i.e., Ca 2+ extrusion through the Na +/ Ca 2+ exchanger operating in forward mode. The cytoskeletal disruption and enlargement of the distal tubules seen in vivo was accompanied by an increase in Na +/K +-ATPase expression and activity, possibly as part of a mechanism by the renal cortex to compensate for renal damage. The natriuretic response observed after treatment with B. alternatus venom could enhance Na +/K +-ATPase expression and activity in post-proximal segments of intact nephron. This increased pump activity could in turn lead to greater sodium reabsorption in the post-proximal segments.
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A
B
C
D
E
F
Fig. 9. Expression of the α1 subunit of Na+/K+-ATPase assessed by immunofluorescence in renal cortex in saline-treated rats (controls) (A,B) and 6 h (C,D) and 24 h (E,F) after the injection of B. alternatus venom. Note the intense staining for the α1 subunit of Na+/K+-ATPase after 6 h (C,D) compared to control tissue (A,B). Twenty-four hours after venom (E,F), the staining was less intense than after 6 h, but was still greater than in control tissue (A,B). Panels (C) and (E) are the same magnification as (A), and (D) and (F) are the same magnification as (B). Bars: 30 μm.
Based on these considerations, we suggest that the enhanced Na +/K +-ATPase expression and activity seen after the injection of B. alternatus venom may be a protective mechanism aimed at preserving renal function during acute renal damage. This conclusion agrees with Tomaz et al. [59] who found that inhibition of Na+–K +-ATPase activity with ouabain potentiated the myonecrosis (measured as creatine kinase release) caused by B. jararacussu venom in mouse skeletal muscle in vitro, i.e., normal functioning of the enzyme attenuated myonecrosis. As noted above, rats injected with B. alternatus venom showed signs of renal failure such as proteinuria and reduced creatinine clearance 6–48 h post-venom. The time scale of these changes in renal function agrees with the onset of renal dysfunction seen in clinical studies of Bothrops bites in which renal failure occurs 18 h to 5 days after envenoming (within 24 h in 56% of patients) [8,9]. Although the high number of variables associated with envenoming in humans (patient and snake age, amount of venom injected, site of bite, first aid measures used, time to hospital admission, use of antivenom) makes it difficult to directly compare clinical cases with controlled experimental studies, it is nevertheless interesting to note that the alterations in Na +/K +-ATPase expression and activity seen here
(within 24 h) coincided with the changes in renal function seen experimentally and clinically. In conclusion, B. alternatus venom causes significant morphological and functional changes in rat renal tissue, with enhanced Na+/K+-ATPase expression and activity. This increase in Na+/K +-ATPase expression and activity may attenuate renal dysfunction during venom-induced damage.
Role of funding source None of the funding agencies indicated in the acknowledgements was involved in the study design, data collection, analysis and interpretation, or in the preparation and writing of the article and the decision to submit the paper for publication.
Conflict of interest statement None of the authors have any conflicts of interest related to this work.
A. Linardi et al. / Biochimica et Biophysica Acta 1810 (2011) 895–906
Acknowledgements The authors thank José Ilton dos Santos, Maiara Daguana and Marta Beatriz Leonardo for technical assistance, and Dr. Maria Alice da Cruz-Höfling (Departamento de Histologia e Embriologia, IB, UNICAMP) for providing laboratory space and advice in the early stages of this work. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). K.C.C. and T.A.A.R.S. were supported by doctoral studentships and A.L. and E.H.M. were supported by post-doctoral fellowships from FAPESP. S.H. is supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References [1] F.O.S. França, C.M.S. Málaque, Acidente botrópico, in: J.L.C. Cardoso, F.O.S. França, F.H. Wen, C.M.S. Málaque, V. Haddad Jr. (Eds.), Animais Peçonhentos no Brasil: Biologia, Clínica e Terapêutica dos Acidentes, Sarvier/FAPESP, São Paulo, 2003, pp. 72–86. [2] D.A. 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