Purification and renal effects of phospholipase A2 isolated from Bothrops insularis venom

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Toxicon 51 (2008) 181–190 www.elsevier.com/locate/toxicon

Purification and renal effects of phospholipase A2 isolated from Bothrops insularis venom$ Marcus Davis Machado Bragaa, Alice Maria Costa Martinsb, Claudeˆnio Dio´genes Alvesb, Dalgimar Beserra de Menezesa, Rene´ Duarte Martinsc, Paulo Se´rgio Ferreira Barbosac, Isadora Maria de Sousa Oliveirac, Marcos Hikari Toyamad, Daniela Oliveira Toyamae, Eduardo Brito dos Santos Diz Filhod, Fabio Henrique Ramos Fagundesd, Manasse´s Claudino Fontelese, Helena Serra Azul Monteiroc, a Department of Pathology, Federal University of Ceara, Fortaleza, Ceara´, Brazil Department of Clinical and Toxicological Analyses, Federal University of Ceara, Fortaleza, Ceara´, Brazil c Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, Fortaleza, Ceara´, Brazil d Sa˜o Vicente Unity, Campus of Litoral Paulista, Paulista State University (UNESP), Sa˜o Paulo, Brazil e Biological Science, Exact and Experimental Faculty, Presbiterian Mackenzie University, Sa˜o Paulo, Brazil b

Received 30 May 2007; received in revised form 21 August 2007; accepted 31 August 2007 Available online 11 September 2007

Abstract Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A2 protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A2 (PLA2) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA2 of Bothrops jararaca and Bothrops asper. Phospholipase A2 isolated from B. insularis (Bi PLA2 ) venom (10 mg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n ¼ 6). Bi PLA2 increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and

$ Ethical statement: The experiments follow the methodology recommended by the international ethical standards of the scientific committee of our university (Comitteˆ de E`tica e Pesquisa com Animais). Corresponding author. Unidade de Pesquisas Clı´ nicas/UFC, Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceara´, CP 3229 Fortaleza, Ce 60420-970, Brazil. Tel.: +55 85 3223 6982; fax: +55 85 32815212. E-mail addresses: [email protected], [email protected] (H.S. Azul Monteiro).

0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.08.017

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chloride tubular reabsorption (%TCl) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA2 isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bothrops insularis; Phospholipase A2; Renal biological activity

1. Introduction Bothrops insularis is a snake known in Brazil as Jararaca ilhoa, found only in an island of which the fauna is composed predominantly of birds, the Queimada Grande Island of the coast of the state of Sa˜o Paulo (Cogo et al., 1993). Various serpents possess phospholipase A2 (PLA2) in their venom (Bon, 1997). The nonneurotoxic phospholipase A2 myotoxins are very common and abundant in crotalids and bothropics venoms (Gutierrez et al., 1986; Rosenberg, 1990; Soares et al., 2000). There are 3 known types of phospholipases isolated from venoms of serpents, the classic one, with aspartic acid in carbon 49, which catalyses the hydrolysis of ester connected to the position sn-2 of glycerophospholipid (Asp49), and variants, also denominated PLA2-like proteins for their structural similarity, containing lysine in place of aspartic acid (Lys49), (Lomonte et al., 2003) or with serine occupying position 49 (Ser49) (Krizaj et al., 1991; Polga´r et al., 1996), these latter two being enzymatically inactive (Lomonte et al., 2003). In vivo effects consist in promotion of degranulation of mast cells with consequent increase in vascular permeability and formation of oedema (Landucci et al., 1998); myotoxic, cardiotoxic, cytotoxic, hypotensive and pro-inflammatory effects, as well as effects on coagulation and platelet activation, were also demonstrated (Valentin and Lambeau, 2000), besides the already known renal changes in the perfused isolated rat kidney system (Barbosa et al., 2002). Snake venoms produce serious kidney complications in snake bite victims, especially acute renal failure (Amaral et al., 1986; Rezende, 1989). The pathogenesis of the renal alterations following envenomation by Bothrops species is not well defined so far and appears to be multifactorial (Nancy et al., 1991); it could be related to the addictive or synergistic effects of different toxins and enzymes present in the venoms (Ferreira et al., 1992). To further evaluate the renal effects of PLA2 isolated from the B. insularis venom, its nephro-

toxicity was studied through the model of isolated perfused rat kidney. 2. Material and methods 2.1. Purification of PLA2 protein from B. insularis venom. Samples of B. insularis venom were provided by Butantan Institute. A dose of 25 mg was dissolved in 0.3 M of ammonium bicarbonate, at pH 7.9, and clarified by centrifugation at 4500g for 2 min. The supernatant was applied and fractionated in a column (1  60 cm) of Superdex 75 (Pharmacia) that was pre-incubated with the same buffer used for the dissolution of the venom. The flow rate was 0.2 mL/min and the elution profile was monitored at 280 nm of absorbance. During the chromatographic run aliquots of 30 mL were collected and subjected to the enzymatic assay conducted as described in 2.1.1 and 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) used as substrate for PLA2. After chromatography, the fractions were lyophilized and myonecrosis evaluated by detection of creatine kinase levels, which were monitored by the aliquots of each fraction. The main myonecrotic PLA2 fraction (7.5 mg) was dissolved in 400 mL of ammonium bicarbonate buffer 0.05 M, pH 7.8, until complete dissolution. The sample was clarified at high-speed centrifugation and the supernatant was applied on the Protein Pack SP 5PW previously equilibrated with the same buffer used for venom dissolution. The proteins were eluted at constant flow rate of 1.0 mL/min, in a linear gradient (0.05–1.0 M) of ammonium bicarbonate and the elution profile was monitored at 280 nm. Fractions containing PLA2 myotoxins were pooled, lyophilized and stored at 40 1C. Molecular mass and homogeneity of the eluted protein were evaluated by reverse-phase high-performance liquid chromatography (HPLC) (0.1  30 cm column of m-Bondapack C18, Waters) using a linear and discontinuous gradient 0–100% of acetonitrile in 0.1% trifluoroacetic acid (v/v). Three milligrams of myotoxic PLA2 protein from the ion exchange was then

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dissolved in 250 mL of buffer A and centrifuged at 4500g for 2 min and the supernatant was applied on the analytical reverse-phase HPLC, previously equilibrated with buffer A (0.1% trifluoroacetic acetic acid (TFA)) for 15 min. The elution of the protein was then conducted by means of a linear gradient of buffer B (66.6% acetonitrile in buffer A) and the chromatographic run was monitored at 280 nm of absorbance. After elution the fraction was lyophilized and stored at 40 1C. The purity degree of phospholipase enzyme was assayed through two-dimensional (2D) electrophoresis and MALDI-TOF mass spectrometry. 2D electrophoresis was conducted as described by Anderson (1991). The grade of purity of the protein obtained by means of reverse-phase HPLC was evaluated by MALDI-TOFF mass spectrometry. 2.1.1. N-terminal sequence Ten milligrams of purified protein was dissolved in 200 mL of 6 M guanidine chloride (Merck, Darmstadt, Germany) containing 0.4 mM Tris–HCl and 2 mM EDTA (final pH 8.15). Nitrogen was flushed over the top of the protein solution for 15 min, then reduced with DTT (6 M, 200 mL) and carboxymethylated with 14C-iodoacetic acid and cold iodoacetic acid. Nitrogen was again flushed over the surface of the solution and the reaction tube sealed. The solution was incubated in the dark at 37 1C for 1 h and desalting was done on a Sephadex G-25 column (0.7  12 cm) in 1 mM acetic acid buffer. The eluted protein, then reduced and carboxymethylated (RC), was lyophilized and stored at 20 1C. Analysis of the amino acid sequence of the RC-protein was performed with an Applied Biosystems model Procise f gas–liquid protein sequencer. The phenylthiohydantoin (PTH) derivatives of the amino acids were identified with an Applied Biosystems model 450 microgradient PTH analyser. 2.1.2. Measurement of PLA2 activity PLA2 activity was measured by the technique described by Holzer and Mackessy (1996), modified to 96-ELISA well plates. The standard assay mixture contained 200 mL of buffer (10 mM Tris– HCl, 10 mM CaCl2 and 100 mM NaCl, pH 8.0), 20 mL of substrate (4N3OBA), 20 mL of water and 20 mL of PLA2 in a final volume of 260 mL. After the addition of PLA2 (20 mg), the mixture was incubated for up to 40 min at 37 1C, the absorbance being read at 10 min intervals. Enzyme activity, expressed as

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the initial velocity of the reaction (Vo), was calculated based on the increase in absorbance after 20 min. All assays were done in triplicate and the absorbances at 425 nm were measured by a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). 2.1.3. Myotoxic activity The CK-UV kinetic kit from Sigma was utilized to assay creatine kinase (CK). The myotoxins (1 mg/ mL) were injected i.m. (50 mL) into the left gastrocnemius muscle of male Wistar rats (110–120 g; n ¼ 6). Control rats received an equal volume of 0.15 M NaCl. After 3 h, the rats were anaesthetized and blood was collected from the abdominal vena cava into tubes containing heparin as anticoagulant. The plasma was stored at 4 1C for a maximum of 12 h before assaying. The amount of CK was then determined in 4 mL of plasma incubated for 3 min at 37 1C with 1.0 mL of the reagent according to the Sigma kinetic CK-UV protocol. Activity was expressed in U/L, one unit resulting from the phosphorylation of 1 mmol of creatine/min at 25 1C. 2.2. Perfused kidney assay Adult male Wistar rats weighing 250–300 g were fasted 24 h with water ad libitum before each experiment and anaesthetized with sodium pentobarbital (50 mg/kg body weight) following ethical guidelines approved by the local committee. The perfusate was a modified Krebs–Henseleit solution (MKHS) containing in mmol/L: 118 NaCl, 1.2 KCl, 1.18 KH2PO4, 1.18 MgSO47H2O, 2.5 CaCl22H2O, and 25 NaHCO3 and 6 g of bovine serum albumin (BSA) was added to 100 mL of MKHS and dialyzed for 48 h at 4 1C against 10 volumes of MKHS. Immediately before the beginning of each perfusion we added 50 mg of urea, 50 mg of inulin and 100 mg of glucose to a final perfusate volume of 100 mL. The pH was then adjusted to 7.4 and the solution placed into the perfusion system. The perfusion model followed the method described by Fonteles et al. (1983). The perfusion pressure was measured at the tip of the stainless-steel cannula and was allowed to fluctuate under experimental conditions, being carefully kept at 120–140 mmHg during the internal control period of 30 min. In each experiment, the recirculating perfusion system employed 100 mL of MKHS and lasted 120 min. After an equilibration period of 15–20 min the experiments

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were started. Perfusion pressure was measured at 5 min intervals. Every 10 min, samples of urine obtained by the cannulated right ureter and perfusate were collected for further analysis of sodium, potassium, inulin and osmolality. Sodium and potassium were measured by flame photometry, and inulin was determined by direct hydrolysis as described by Walser et al. (1955) and modified by Fonteles et al. (1983). Osmolality was measured in a vapour pressure osmometer (WESCOR 5100c vapor pressure). Chloride analysis was carried out using a LabTest kit. PLA2 from the venom of B.

insularis (10 mg/mL) was always added to the system 30 min after the beginning of each experiment (n ¼ 6). Data were measured at 10 min intervals and averaged every 30 min at 30, 60, 90 and 120 min. 2.2.1. Histological evaluation After the experiment of renal perfusion, both kidneys were removed and fixed in 10% formaldehyde for histological processing. Kidney tissue was embedded in paraffin, cut into 3–5 mm sections, stained with haematoxilin and eosin and processed further for light microscopy.

Fig. 1. Chromatographic profile of Bothrops insularis whole venom disclosed VII main fractions designated as I, II, III, IV, V, VI and VII and the main PLA2 enzymatic activity was detected in fractions IV and V (a). The chromatographic profile also showed that fraction IV is the main fraction eluted in the molecular exclusion column. This fraction was subjected to a new chromatographic procedure on the ion exchange chromatography that permitted us to elute 5 main fractions named IV-1 to IV-5 and IV-4 was the main fraction eluted from the column (b). The molecular homogeneity of this fraction was characterized by analytical reverse-phase HPLC (c).

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2.2.2. Statistical analysis The results were expressed as means7SEM (n ¼ 6). Statistical evaluation was determined by analysis of variance (ANOVA) and corrected by the Bonferroni test. Statistical significance was set at 5%. 3. Results 3.1. Purification of PLA2 protein from B. insularis venom Chromatographic profile of B. insularis whole venom disclosed 7 main fractions designated as I, II, III, IV, V, VI and VII. The main PLA2 enzymatic activity was detected in fractions IV and V (Fig. 1a). The chromatographic profile showed that fraction IV is the main fraction eluted in the molecular exclusion column. This fraction was then subjected to a new chromatographic procedure on the ion exchange chromatography that allowed us to elute 5 main fractions named IV-1 to IV-5, IV-4 being the main fraction eluted from the column (Fig. 1b). The molecular homogeneity of this fraction was characterized by analytical reverse-phase HPLC (Fig. 1c). The molecular homogeneity of this PLA2 as demonstrated by MS showed a molecular mass of 13984.20 Da and its N-terminal sequence revealed high amino acid identity (95%) with Bothrops jararaca and Bothrops asper PLA2. The two-dimensional electrophoresis of the Bi PLA2 disclosed only one protein spot with an estimated molecular mass of approximately 14 kDa and experimental pI ca. 8.6 (Fig. 2). Bi PLA2 also showed high amino acid sequence identity with other typical myotoxic PLA2; nevertheless, Bi PLA2 showed some amino acid replacement such as L(4) for F(4), Q(11) for K(11) and N(27) for Y(27) (Fig. 3). Fractions III and IV displayed the highest CK levels (Fig. 4a); Fraction IV (Bi PLA2), despite some amino acid replacement, when compared to other classical PLA2 myotoxins, such as BthTx-I and Bi PLA2, indeed showed myonecrotic properties (Fig. 4b). 3.2. Perfused kidney assay The group treated with phospholipase A2 had an increase in perfusion pressure (Fig. 5a), renal vascular resistance (RVR) (Fig. 5b), urinary flow (UF) (Fig. 6a) and in glomerular filtration rate (GFR) (Fig. 6b). Sodium (Fig. 7a) and chloride (Fig. 7b) tubular transport was reduced at 120 min

Fig. 2. Panel a shows the two-dimensional electrophoresis of 125 mg of Bothrops insularis whole venom and panel b shows the two-dimensional electrophoresis of Bi PLA2 (50 mg).

of perfusion, but there was no significant change in potassium transport (Table 1). 3.3. Histological alterations In the control group, the kidneys showed sparse areas of hydropic vacuolar degeneration of the tubular lining cells, mild intratubular deposition of proteic material; glomeruli, interstitium and vessels showed no abnormalities. The group treated with B. insularis phospholipase A2 presented mild to moderate focal acute tubular necrosis in the convoluted proximal tubules; the nuclei of some proximal tubular cells presented pyknosis with clumping of chromatin (Fig. 8).

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Fig. 3. (a) Molecular homogeneity of this PLA2 was demonstrated by MS that showed a molecular mass of 13984.20 Da. (b) N-terminal region showed high amino acid sequence homology (95%) with the ones of Bothrops jararaca and Bothrops asper neurotoxic PLA2. Also, Bi PLA2 showed high amino acid sequence homology with other typical myotoxic PLA2, but Bi PLA2 displayed some amino acid substitution such as L(4) for F(4), Q(11) for K(11), N(27) for Y(27).

Tubular epithelial cells presented cytoplasmic vacuolation, irregular profiles of the brush border and some desquamation of cells to the lumina; proteinaceous deposits were observed within the proximal and distal tubules (Fig. 9); 46.61% out of 118 glomeruli studied under light microscopy by 10  low-power field, showed significant amount of a proteinaceous deposit in the Bowman space.

4. Discussion B. insularis phospholipase A2 showed enzymatic activity on the synthetic chromogenic substrate for PLA2 and some myonecrotic activity. The triad of amino acids constituted by F(5)/A(102)/F(106) is a common feature found for Asp-49 myotoxic PLA2, although in the case of Bi PLA2 we only found F(5)

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Control

150 Perfusion Pressure (mmHg)

187

*

*

*

Bi PLA2

100

50

0 30

60

90

120

Time (minutes) Control

7.5

*

*

*

Renal Vascuar Resistance (mmHg / mL.g-1.min-1)

Bi PLA2

5.0

2.5

0.0 Fig. 4. (a) Fractions III and IV revealed the highest CK levels. (b) Bi-IV, despite the replacement of some amino acids, when compared to other classical myotoxic PLA2, such as BthTx-I and Bi PLA2, showed myonecrotic properties.

in its N-terminal region. Asp49 PLA2 may lead to myonecrosis via several mechanisms which are not completely dependent of enzymatic activity. Most venoms probably exert their effects on cells and tissues by means of pharmacological proprieties determined by several biologically active components (Monteiro et al., 2001). Barbosa et al. (2002) obtained similar results with the use of the fraction PLA2 isolated from B. moojeni venom. PLA2 from B. moojeni venom caused an increase in PP, RVR, FU, GFR, and a decrease in the transport of Na+, contrary to the total venom, which provoked a decrease in the renal vascular parameters, and an increase in UF and GFR. It is proposed that the C-terminal region rich in lysine of this PLA2, would be an inducer of the toxic activity by direct aggression to the cellular

30

60

90

120

Time (minutes) Fig. 5. Effects of Bothrops insularis phospholipase (Bi PLA2; 10 mg/mL) on vascular parameters. (a) Perfusion pressure (PP) (mmHg); (b) renal vascular resistance (RVR) (mmHg/mL/g/min). Data are expressed as means7SEM from 6 different animals. Statistical analyses were carried out by ANOVA, comparing with a control group in which the kidneys were perfused with only MKHS with *po0.05.

membrane, stimulating the release of vasoconstrictors, independent of the activity of enzymes as occurs with many PLA2 of snake venoms with Lys49 (Lomonte et al., 2003). Barbosa et al. (2005) demonstrated that Bothrops jararacussu myotoxin I (Lys 49) and B. jararacussu myotoxin II (Asp 49) purified from B. jararacussu presented antimicrobial activity and produced similar alterations in renal physiology, such as increased perfusion pressure, renal vascular resistance, diuresis, natriuresis and kaliuresis. Indomethacin was able to reverse the renal effects induced by B. jararacussu myotoxin I

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Control Bi PLA2

0.2

*

*

*

0.1

% TubularTransport of Na+

Urinary Flow (mL.g-1.min-1)

0.3

Control

100

Bi PLA2 *

75 50 25 0

0.0 30

60

90

30

120

60

Glomerular Filtration Rate (mL.g-1.min-1)

Control

*

Bi PLA2

*

1.0

0.5

0.0

%% Tubular Transport of Cl-

Time (minutes) 1.5

90

120

Time (minutes)

Control

100

Bi PLA2 *

75 50 25 0

30

60

90

120

Time (minutes) Fig. 6. Effects of Bothrops insularis phospholipase (Bi PLA2) (10 mg/mL) on urinary parameters. (a) Urinary flow (UF) (mL/g/ min); (b) glomerular filtration rate (GFR) (mL/g/min). Data are expressed as means7SEM from 6 different animals. Statistical analyses were done by ANOVA, comparing with a control group in which the kidneys were perfused with only MKHS with *po0.05.

(BthTx I), and to reduce the effects promoted by B. jararacussu myotoxin II (BthTx II), which suggests the presence of eicosanoids as possible mediators for the renal effects. On the other hand, other pharmacological sites could be involved in this activity independent of the enzymatic activity of BthTx II. The PLA2 myotoxin isolated from B. insularis venom presented enzymatic activity similar to BthTx II and produced similar alterations in renal physiology such as increases in PP, RVR, UF and a GFR, and decrease in the transport of Na+ and Cl. Histopathological examination disclosed the presence of an eosinophilic proteinaceous material in the Bowman space possibly due to extra leakage of

30

60

90

120

Time (minutes) Fig. 7. Effects of Bothrops insularis phospholipase (Bi PLA2) (10 mg/mL) on the percentage of sodium tubular transport (% TNa+). (a) Percentage of chloride tubular transport (% TCl). (b) Data are expressed as means7SEM from 6 different animals. Statistical analyses were done by ANOVA, comparing with a control group in which kidneys were perfused with only MKHS with *po0.05.

perfusion fluid through the glomerular capillaries. Chacur et al. (2003) reported the capacity of phospholipase A2, especially Asp-49 PLA2, of inducing oedema in rat paws through the release of bradykinin. They also supposed that this effect involves the participation of biogenic amines, cytokines and prostanoids that potentiate bradykinin activity. Thus, Asp-49 PLA2, used in our study, probably produced extra leakage of perfusion fluid to the Bowman space; perfusion pressure might also contribute to that extra leakage by increasing hydrostatic pressure. Acute tubular necrosis, as described for many venoms (Boer-Lima et al., 1999), mildly and focally demonstrated in renal cortex, may reflect direct cytotoxic aggression by the phospholipase.

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Table 1 Effects of Bothrops insularis phospholipase A2 (10 mg/mL) on kidney Variables

30 min

60 min

90 min

120 min

%TK+ PhospholipaseA2 Control

66.4373.60 69.1374.14

64.2973.19 69.0475.68

7.8372.77 7.8474.21

71.9372.59 69.9476.86

Results are expressed as means7SEM from 6 different animals for each group. Statistical analyses were done by ANOVA, comparing with a control group in which the kidneys were perfused with only MKHS with *po0.05. All data were averaged between 30 min intervals (30, 60, 90, 120). Phospholipase A2 was always added after the first 30 min of each experiment. %TK+ ¼ percent of potassium tubular transport.

lining cells and desquamation of these cells into the tubular lumina, which might reflect enzymatic activity of PLA2 on the cell membrane phospholipids and production of arachidonic acid derivatives. Some of these findings, though limited to the renal cortex and less intense, were present in the tubular lining cells of the control group. In conclusion, Phospholipase A2 isolated from B. insularis venom promotes renal alterations in the isolated perfused rat kidney. References Fig. 8. Focus of condensed nuclear chromatin representative of moderate acute tubular necrosis (HE) (40  ). Canon, model Power Shot A 95, 5.0 mp.

Fig. 9. Increased proteinaceous deposit within distal and proximal tubules, cell swelling and vacuolization of the proximal tubular lining cells with discontinuity of the brush borders of the proximal tubular lining cells and desquamation of these cells to tubular lumina; glomeruli with extravasation of protein within the Bowman space (HE) (20  ). Canon, model Power Shot A 95, 5.0 mp.

Proximal tubular cells presented hydropic changes throughout the kidney accompanied with discontinuity of the brush borders of the tubular

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