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Evaluation of abrin induced nephrotoxicity by using novel renal injury markers
Accepted Manuscript Evaluation of abrin induced nephrotoxicity by using novel renal injury markers Bhavana Sant, P.V. Lakshmana Rao, D.P. Nagar, S.C. Pant, A.S.B. Bhasker
PII:
S0041-0101(17)30094-6
DOI:
10.1016/j.toxicon.2017.03.007
Reference:
TOXCON 5594
To appear in:
Toxicon
Received Date: 25 August 2016 Revised Date:
8 March 2017
Accepted Date: 9 March 2017
Please cite this article as: Sant, B., Rao, P.V.L., Nagar, D.P., Pant, S.C., Bhasker, A.S.B., Evaluation of abrin induced nephrotoxicity by using novel renal injury markers, Toxicon (2017), doi: 10.1016/ j.toxicon.2017.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of abrin induced nephrotoxicity by using novel renal injury markers
2 Bhavana Sant, P.V. Lakshmana Rao, D.P. Nagar, S.C. Pant and A. S. B. Bhasker*
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Division of Pharmacology and Toxicology, Defence Research and Development Establishment,
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Jhansi Road, Gwalior 474002, India.
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*Corresponding Author:
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Tel: +91-751-2233495 Fax: +91-751-2341148
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E-mail: [email protected]
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Abstract
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Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus
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precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes
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cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study
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we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress,
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inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed
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to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-
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toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were
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observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as
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observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by
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using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney
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injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity
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of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due
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to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of
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abrin.
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1. INTRODUCTION
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Abrin and ricin are among the most toxic plant proteins (Roxas-Duncan and Smith, 2012). Both
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proteins are derived from the seeds of the plants Abrus precatorius and Ricinus communis
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respectively. Abrin shows significant similarities to ricin both at the sequence and structural
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levels; however abrin is a more potent toxin than ricin (Surendranath and Karande, 2008). Both
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toxins belong to the family of type II Ribosome Inactivating Proteins (RIPs). Toxins from the
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RIP family irreversibly inactivate protein synthesis through an enzymatic mechanism. The
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reported human fatal dose of abrin is 0.1-1µg/kg of body weight, with a molecular mass of
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approximately 60-65KDa (Wooten et al., 2014). Abrin is a protein synthesis inhibitor and
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induces apoptosis in cells (Mishra and Karande, 2014). Abrin is a heterodimer consisting of two
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disulfide-linked polypeptides, known as the A-chain and B-chain. The active A-chain moiety has
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an enzymatic function. The toxicity of A-chain is due to its RNA N-glycosidase activity by
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which it brings about depurination of adenine at 4324 position in the 28S ribosomal RNA. The
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end result of this activity is complete inhibition of protein synthesis leading to cell death (Olsnes,
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2004). The B chain has galactose-binding capability and shows lectin type activity, helping the
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toxin to internalize inside the cell.
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subsequently at higher doses, severe necrosis in the organs of poisoned animals and in cultured
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cells (Griffiths et al., 1987; Narayanan et al., 2004). Earlier, we investigated oxidative stress
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mediated DNA damage and cell death by using ricin and abrin in vitro (Rao et al., 2005; Bhaskar
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et al., 2008).
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Accidental or intentional exposure to abrin has been frequently reported (Sahni et al., 2007;
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Sahoo et al., 2008). Many of these poisoning cases showed unusual symptoms like central
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nervous system toxicity and renal failure (Subramanyam, 2008).
At cellular level both abrin and ricin cause apoptosis and
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Nephrotoxicity is an important target of toxicological studies due to its crucial role in drug
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excretion and detoxification. A number of studies have described neuroinflammation and
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damage caused by nephrotoxicity (Burn and Bates, 1998; Liu et al., 2008; Brouns and Deyn,
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2004). Various nephrotoxicants produce different types of toxicity syndromes, including acute
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renal failure, chronic renal failure, renal tubular defects, nephrotic syndromes and hypertension.
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Tubular kidney cells are particularly vulnerable to toxin mediated acute kidney injury due to
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their disproportionate exposure to circulating chemicals and transport processes that result in
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high intracellular concentration. Due to abundant presence of polyunsaturated fatty acids in the
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composition of renal lipids, kidneys are very susceptible to oxidative stress (Ozbek, 2012). RIPs
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like trichosanthin and shiga toxin have been reported to cause kidney injury via tubular damage
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(Tang et al., 1997; Porubsky et al., 2014). Ricin induced nephrotoxicity is characterized by
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altered serum biomarkers of kidney function, increased lipid peroxidation and decreased activity
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of antioxidant enzymes, leading to oxidative stress induced renal toxicity (Kumar et al., 2003).
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Oxidative stress, which occurs when there are excessive free radical production or low
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antioxidant levels, has been reported in chronic kidney disease conditions. These free radicals
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can damage proteins, lipids, carbohydrates and nucleic acids. Oxidative stress leads to tissue
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damage through different mechanisms, including lipid peroxidation, DNA damage and protein
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modification. These mechanisms have been implicated in the pathogenesis of several systemic
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diseases including kidney disease (Ozbek, 2012). Glutathione serves to protect cells from
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oxidative stress. Glutathione, normally present in high amounts in tubular cells, can neutralize
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ROS. Reduced kidney cellular glutathione levels and depletion of other antioxidants lead to
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oxidative stress in cells (Abraham et al., 2013). Inflammation is now believed to play a major
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role in the pathophysiology of acute kidney injury. Leukocytes and renal tubular cells release
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many cytokines into the injured kidney and are important components of both the initiation and
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extension of inflammation during acute kidney injury (Akcayet et al., 2009). TNF-α mediated
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kidney injury has also been reported to follow exposure to shiga toxin, a known RIP of bacterial
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origin (Lentz et al., 2010).
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Oxidative stress may increase the production of TNF-α and other cytokines by activation
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of NFκB (Manna et al., 1998). TNF-α is a proinflammatory marker produced by immune cells
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like macrophages and lymphocytes; however, further studies revealed that it is also produced by
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endothelial and epithelial cells (Ramseyer and Garvin, 2013). Recruitment of immune cells into
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the kidney, which releases cytokines, may cause inflammation and cell death (Zhu et al., 2009).
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TNF-α is a strong inducer of matrix metalloproteinase, MMP-9 (Baharet et al., 2010). Zhou et al.
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(2009) described TNF-α induced expression of MMP-9 mediated by activation of p21-activated-
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kinase-1 and JNK pathway. Matrix metalloproteinases (MMPs) are enzymes responsible for
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degradation of the extracellular matrix (ECM) and are involved in the pathogenesis of ischemia-
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re-perfusion injury. MMP‑9 is gelatinase and cleaves to denatured collagens (gelatins) and
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laminin, as well as certain chemokines. In the kidney, MMP’s are synthesized by intrinsic
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glomerular cells and tubular epithelial cells. ECM degrading ability of MMPs play an important
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role in the progression of nephropathies. Altered MMP expression, like ability of MMP-2 to
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induce or sustain an inflammatory mesangial cell phenotype, has been observed in a large
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number of kidney diseases (Marti, 2000).
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Some of the prominent biomarkers of kidney injury include NGAL (Neutrophil Gelatinase
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Associated Lipocalin), Cystatin C and Clusterin. (Fassett et al., 2011). NGAL, a ubiquitous 25
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KDa protein, covalently bound to gelatinase from human neutrophils, is a marker of tubular
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injury (Cowland and Borregaard, 1997; Dharnidharka et al., 2002). Serum and urine NGAL
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levels predict acute kidney injury in different clinical conditions (Mishra et al., 2005). Cystatin C
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is produced in all nucleated cells. This protein is a marker of kidney injury from drug toxicity
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and many chronic diseases. It is a marker of glomerular filtration rate and also indicates proximal
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tubule injury. Clusterin is a multifunctional glycoprotein with roles in metabolism and transport
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of lipids. Reduced clusterin expression results in an increase in cell death or renal tissue injury.
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The mechanism behind clusterin’s kidney protection is unclear. Up regulation of clusterin
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expression may cause anti-apoptosis and inactivation of complements which may partly
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contribute in protection of kidney injury (Zhou et al., 2010).
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The renal response to poisons is dynamic, and the kidney adapts to maintain homeostasis during
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the cascade of repair and recovery that follows the primary insult (Bach,1989). There are reports
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on abrin-induced neurotoxicity (Bhasker et al., 2014) and hepatotoxicity, but little information is
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available about the nephrotoxicity potential of abrin. Hence, the objectives of this study were to
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determine the following:
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a) The mechanism of acute kidney injury after abrin exposure in a mouse model by using
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different kidney specific markers and histopathology.
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b) The role of inflammation, proximal tubular damage, oxidative stress, and vascular injury on
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the pathogenesis of abrin-induced nephrotoxicity.
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2. MATERIALS AND METHODS
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2.1 Animals
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The study used BALB/c mice randomly bred in the Institute’s animal facility, weighing between
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23 and 27 g. The animals were maintained according to standard conditions of temperature and
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humidity (25±20C, relative humidity 40-60%), and the animals were fed a standard pellet diet
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(Ashirwad Brand, Chandigarh, India). Food and water were given ad libitum. The animals were
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handled according to the guidelines of CPCSEA (Committee for the Purpose of Control and
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Supervision on Experiments on Animals). The study was approved by Institutional Animal
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Ethics Committee, a statutory committee constituted by CPCSEA, Animal Welfare Cell,
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Ministry of Environment, Forests and Climate Change, Government of India.
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2.2 Chemicals
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Abrin was isolated from A. precatorius seeds as described elsewhere (Kumar et al., 2008). The
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purified abrin was lyophilized and stored at −80 ◦C and reconstituted as and when required by
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PBS. Reduced glutathione (GSH) was from Across (Belgium). O-phthaldialdehyde (OPT) was
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from Fluka (USA). All other chemicals were of extra pure grade and obtained from Sigma
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Chemical Co. (St. Louis, USA) unless otherwise mentioned.
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2.3 Experimental design
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The mice were divided into six groups: Control, 0.5LD50 1, 3 and 7 day, and 1LD50 1 and 3
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day. Because not all animals survived to 7 days in the 1LD50 dose group, data for 7 day is not
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included in the data analysis. All treated groups including control consisted of six animals each.
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Four animals were used for biochemical assays and two for performing histopathology. 0.5LD50
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(0.88µg/kg) and 1LD50 (1.76µg/kg) dose of abrin toxin was administered intraperitoneally in
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mice. Control mice received an equal volume PBS by same route. Mice were handled in
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accordance with the standard guide for the care and use of laboratory animals. At the end of
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experimental period, animals were sacrificed by cervical dislocation under deep anesthesia.
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Following induction of anesthesia, the mice were subjected to a midline abdominal incision and
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both left and right kidney tissues were harvested.
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2.4 Biochemical assays
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GSH level was measured spectrofluorimetrically using the method of Hissin and Hilf (1976) and
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expressed as µmloes of GSH/ g tissue. Malondialdehyde (MDA) content was evaluated using the
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thiobarbituric acid (TBA) (Ohkawa et al., 1979). Absorbance was measured at 532 nm to
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determine the MDA content. The level of lipid peroxidation in kidneys is expressed as nmoles of
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MDA/g tissue.
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2.5 Western blot analysis of TNF-α, Clusterin, Cystatin C, NGAL.
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Polyclonal anti-TNF-α, anti-Clusterin, and anti-NGAL antibodies were purchased from Santa
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Cruz Biotechnology Inc. Monoclonal anti- Cystatin C was purchased from Abcam (UK). Total
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protein was estimated using a Bio-Rad kit, separated by SDS‑PAGE and transferred to
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nitrocellulose membranes. The membrane was treated with a blocking solution (5% skimmed
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milk in TBST) and incubated overnight at 4°C in shaking condition, with various primary
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antibodies at specified dilutions TNF-α (1:500), Clusterin (1:500), NGAL (1: 1000), Cystatin C
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(1:10,000). The membranes were washed four times in TBST for 7 minutes each, followed by
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incubation for 2 hours in horseradish peroxidase-conjugated anti-rabbit and anti-goat secondary
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antibody (1:20000). Membranes were washed again and immunoreactive proteins were
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visualized
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(ProteoQwestTMChemiluminescent Western blotting kit, Sigma-Aldrich), and images were taken
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on a Fusion FX5 image analyser. After western blot analysis, each band was analyzed using
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using
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ImageJ software for quantitative measurement. In short, a constant unit area was selected and
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average density of the total band area of the respective proteins in control and treated groups
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were measured and represented graphically.
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2.6 Protein carbonyl assay
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Carbonylated proteins of kidney tissues were detected using Oxyblot™ Protein Oxidation
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Detection Kit (Chemicon/Millipore). Briefly, the protein samples were prepared and carbonyl
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groups were derivatized using 1x DNPH solution provided in the kit, then the samples were
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resolved on SDS-PAGE, transferred on to nitrocellulose membrane. DNP-derivatized proteins
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were identified by chemiluminescent method using an anti-DNP antibody. Carbonylated protein
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bands were quantified by densitometry using NIH ImageJ software.
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2.7 Substrate gel zymography
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The extracellular activities of MMP-9 in tissue and serum were assessed using gelatin
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zymography. Tissue was homogenized in a lysis buffer containing protease inhibitors. The
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homogenate was centrifuged for 30 min at 10,000x rpm, and supernatant was used as protein
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lysate. Protein content was estimated using Bio-Rad kit, and 40µg of protein was resolved in 8%
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non-reducing SDS electrophoresis gel containing 0.4% gelatin. The gel was washed with
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washing buffer (50 mM Tris–HCl pH 7.5, 2.5% Triton X-100) for 2 h at room temperature and
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incubated overnight in developing buffer (50 mM Tris–HCl pH 7.5, 10 mM CaCl2, 1 M ZnCl2)
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at 370C. Protein bands were visualized using Coomassie blue staining followed by destaining.
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Proteolytic activities were quantified by performing densitometry using NIH ImageJ software
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and results presented graphically.
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2.8 Histopathological examination
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Control and abrin treated animals were sacrificed after specific time points. Tissue samples of
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kidneys were dissected. After fixation small pieces were processed by automated tissue
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processor (Leica TP 1020), dehydrated and embedded in paraffin wax. Multiple sections of 4-5
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µm thick from each block were cut on a rotatory microtome (Microm, Germany), mounted on a
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slide with albumin coating and air dried overnight. The sections were stained with hematoxylin
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and eosin (McMannus and Mowry, 1965) in Leica Autostain-XL. Microscope observation was
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performed under Leica, DMLB microscope and photographs were taken using a DM 500 camera
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(Leica, Germany).
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2.9 Statistical analysis
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One-way ANOVA followed by Dunnet’s test were performed and used for comparison between
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the control and treated groups. The level of significance was set at P ≤ 0.05.
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3. RESULTS
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3.1 Effect of abrin on oxidative stress parameters
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Reduced glutathione, an important intracellular antioxidant, decreased in the kidneys of mice
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treated with 0.5 and 1 LD50 doses of abrin at different time points (Fig. 1). Time dependent
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decrease was found in the 1 LD50 group, and although this pattern was not observed in 0.5LD50
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group, the decrease was still significantly different from the control. On day 1 the GSH levels
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were 1.79 ±0.11, and 1.58 ± 0.20 on day 3. On day 7 GSH levels were 1.77 ± 0.27. Control
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levels of GSH were 3.44 ±0.23. As compared to 0.5LD50 dose, GSH levels were more reduced
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on day 1 and on day 3 of 1LD50 dose (1.37 ± 0.17 & 1.33 ± 0.17). GSH concentration was
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measured in µmoles of GSH/gm of tissue.
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Time dependent increase in lipid peroxidation was observed in the 1LD50, and not in the
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0.5LD50 group (Fig. 2). But MDA concentration in 0.5LD50 group was significantly higher than
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control.
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3.2 Effect on oxidation of kidney proteins and TNF-α
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Protein carbonyl content is not only a biomarker of oxidative stress, but also provides evidence
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of oxidative protein damage. An increased number of oxidized protein bands, which are more
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intense in levels, were detected in a dose and time dependent manner in all the treated groups at
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all the time points compared to control (Fig. 3A-B). Western blot assessment of TNF-α was
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carried out. Time dependent increase in expression of TNF-α was found in 0.5LD50 group as
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compared to control. This increase was also observed in 1LD50 but was not time dependent (Fig.
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4A-B).
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3.3 Kidney and serum zymography
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Zymographic analysis of kidney tissue revealed gelatinolytic activity of pro-MMP-9 and active
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MMP-9 (92 KDa ). Differences were found in the activities of both gelatinolytes as compared to
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control. Increased activity of MMP-9 was observed in abrin treated group (Fig. 5A-B). Serum
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levels of MMP-9 were also analyzed by zymography. At 1LD50 there was time dependent
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increase in MMP-9. A high-molecular-weight complex of NGAL/MMP-9 (125 KDa) was also
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observed in serum. Higher activity of this complex was found at both doses and time points in
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the abrin exposed group compared to control (Fig. 5C). Quantitative analysis through ImageJ
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software is shown in Fig. 5 D-E.
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3.4 Western blot analysis of kidney injury biomarkers
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Neutrophil gelatinase-associated lipocalin (NGAL, also called lipocalin 2 or 24p3), a small 25
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kDa protein that belongs to lopocalin family, is highly expressed during acute renal injury in
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animal models. Protein expression of NGAL was increased in dose and time dependent manner.
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Clusterin is a lipoprotein that has anti-complement effects in membranous nephropathy. Time
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dependent decrease in expression of clusterin was found in the 0.5LD50 group as compared to
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the control. In the 1LD50 group, clusterin decreased more by day 3 than day 1, indicating the
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severity of tubular damage.
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Cystatin C, cysteine protease inhibitor, is a marker for decreased glomerular filtration rate.
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Immunoblot results have shown increased expression of cystatin C in all treated groups as
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compared to the control. Time dependent increase was found in the 0.5LD50 group, but this
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pattern was not seen in the 1LD50 group. No expression was observed in the control group
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(Fig.6A-D). Quantitative analysis of the blots through ImageJ software revealed that the changes
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were significantly different from control (Fig.6E-G).
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3.5 Kidney histology
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The kidney histopathology of mice exposed to abrin sacrificed on days 1, 3 and 7 is shown in
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Fig.7 (a-f). The kidneys of control mice showed normal architecture (Fig. 7a), including normal
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glomerulus, Bowman's space and renal parenchyma. One day after exposure to 0.5 LD50 abrin
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showed negligible degenerative changes. Moderate changes appeared after 3 and 7 days in the
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kidneys of mice treated with same dose, which shows that accumulation of edematous fluid in
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the medullary areas is associated with infiltration of inflammatory cells (Fig. 7c-d). The kidneys
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of mice one day after exposure to1 LD50 abrin showed cloudy swelling in the renal tubule with
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vacuolar degeneration (Fig. 7e). By day 3 they showed severe hemorrhage, glomerular
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congestion, dilatation of the proximal convoluted tubules, obliteration of renal parenchyma, and
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loss of epithelial lining, and the lumen of these tubules was filled with eosinophilic material and
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cell exudate. These changes were associated with infiltration of mononuclear cells (Fig. 7f).
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4. DISCUSSION
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Ribosome inactivating proteins are a group of proteins that share the property of damaging
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ribosomes in an irreversible manner acting catalytically. One of the most potent members of the
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class II RIP family is the phytotoxin abrin produced by the subtropical climber Abrus
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precatorius. Abrin inhibits elongation factor EF-1 and EF-2, preventing protein synthesis and
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leading to cell death (Bradberry, 2004; Robertus, 1991). Due to its high availability and ease of
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preparation, abrin is considered a biological threat, especially in context of bioterrorism. To date,
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there is no established therapeutic countermeasure against abrin intoxication.
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There are some reported cases of abrin poisoning in the USA and India (Roxas-Duncan and
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Smith, 2012), and cases include both accidental and intentional ingestion of seeds. Children are
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at greater risk of ingesting seeds due to their colorful appearance, and the consequences could be
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fatal. After consuming abrus seeds, an 18-month-old child reported with gastrointestinal
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symptoms which were consistent with reported cases of abrin poisoning, but the patient also had
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isolated and significantly elevated alkaline phosphatase levels. Tests could not reveal the
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responsible pathological process (Mazin et al., 2015).
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Abrin is toxic by all routes of exposure, although its lethality depends on its route of
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administration (inhalation, ingestion, or injection) (Goldman et al., 2011). Our laboratory
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recently reported neurodegenerative changes after intraperitoneal abrin exposure in mice
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(Bhasker et al., 2014). Nephrotoxicity of abrin has not been reported before, although Wooten et
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al. (2014) presented a case of abrin poisoning of a 22-month-old girl, who developed a
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remarkable increase in serum kidney markers like creatinine and BUN. In a case report of abrin
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poisoning, a 17-year-old woman showed signs of CNS toxicity and altered sensorium. The
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patient also developed renal failure and an enlarged kidney, hyponatremia (Subrahmanyan et al.,
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2008). Based on above data on abrin poisoning, the present study was designed to evaluate the
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nephrotoxic potential of abrin and possible mechanism mediating nephrotoxicity.
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Reports are available on direct renal injury against other ribosome-inhibiting proteins like shiga
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toxin, which induces apoptosis and TNF-gene expression in the medulla, monocyte/macrophage
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infiltration, platelet aggregation, severe tubular damage and acute deterioration of renal function
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(Yamamoto et al., 2005). Renal impairment reported after infusion of trichosanthin, a type I
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ribosome inactivating protein, resulting in reduction of glomerular filtration rate and tubular
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proteinuria. Both necrotic cell death and apoptosis participated in cell loss from the proximal
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tubules. Such toxicity may be mediated through intracellular events induced by trichosanthin
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(Tang et al., 1997). A report for ricin toxicity has revealed that ricin administered with or without
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lipopolysacharide rapidly produced oliguric renal failure, hemolysis and thrombocytopenia.
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Inflammatory response was also seen with a marked increase in plasma concentrations of
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inflammatory cytokines and a glomerular infiltration of macrophages (Taylor, 1999).
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Several lines of evidence support the hypothesis that ROS are involved in the pathophysiology of
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renal failure (Nath and Norby, 2000). Oxidative stress is seen both systemically and locally at the
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kidney level through the enhancement of ROS production by circulating neutrophils and
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membrane lipid peroxidation in kidney respectively. Carbonylated proteins of abrin exposed
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kidney tissue were detected using OxyBlotTM. Significant increase in oxidized proteins with dose
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and time dependent changes confirms protein oxidation after abrin exposure. Reduction of GSH
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in the kidney contributes to renal epithelial cell death (Gomez et al., 2013). Taken together, our
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results of depleted GSH concentrations and increased MDA content suggests that abrin induces
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oxidative stress and renal epithelial cell damage. Inflammation is one of the manifestations of
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oxidative stress, and the pathways that generate the mediators of inflammation, such as adhesion
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molecules and interleukins, are all induced by oxidative stress.
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Tumor necrosis factor (TNF) alpha is a potent pro-inflammatory cytokine and important
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mediator of inflammatory tissue damage. There are number of reports on TNF-α induced renal
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damage. It may be due in part to recruitment of immune cells into the kidney, releasing cytokines
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that cause inflammation and cell death (Zhu et al., 2009). TNF-α may contribute to abrin
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nephrotoxicity by inciting an inflammatory response within the kidney. It is also likely due to
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direct actions of TNF-α on renal cells. Increased expression and activity of TNF- α after abrin
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exposure directly indicates kidney inflammation. There are several reports detailing TNF-α
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induced expression of matrix metalloproteinases (MMPs). MMPs regulated by various stimuli
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are important in remodeling glomerular ECM, which leads to a number of renal diseases. TNF-α
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was involved in the regulation of MMPs and signal pathways involved in TNF-α-induced MMPs
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expression in rat glomerular mesangial cells (Wang et al., 2014). Expression of MMP-2 and
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MMP-9 has been detected in glomerulus and proximal tubules of rats (Catania et al., 2007).
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There are several reports that support the idea that MMPs mediate acute kidney injury and are
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involved in changes in the glomeruli and tubular epithelial cells (Romero et al., 2009). In the
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present study, our results show TNF-α increased activity of kidney tissue pro-MMP-9 and active
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MMP-9 in the abrin treated group, leading to inflammatory changes. Pro-MMP-9 can also be
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activated in various diseased conditions such as ischemia, a hallmark of acute renal failure. It
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strongly stimulated proteolytic activity of proMMP-9 and MMP-9 in the renal endothelial
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fractions (Caron et al., 2005). NGAL is a biomarker of tubular injury. NGAL protects MMP-9
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from proteolytic degradation and enhances its enzymatic activities by binding and forming the
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MMP-9/NGAL complex. Therefore, NGAL, MMP-9 and their complex MMP-9/NGAL have
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been proposed as biomarkers for numerous malignancies (Carlo, 2013). We found increased
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expression of NGAL/MMP-9 complex in sera of the abrin treated group. Recent data
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demonstrated that increased levels of NGAL are present in chronic kidney disease and acute
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kidney damage (Chakraborty et al., 2012; Bolignano et al., 2008).
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Further evidence for abrin induced kidney damage in the present study was obtained by studying
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other protein markers in the kidney – namely cystatin-C and clusterin – that together offer
332
information on location of injury in the nephron. Cystatin C is a biomarker of the glomerular
333
filtration rate (Cabarkapa, 2015) that is produced in the nucleated cells at a constant amount, and
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its serum concentration does not depend on muscle mass or protein intake. The catabolism of
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cystatin C is mostly done in the kidneys. Our results have shown increased expression of cystatin
336
C which indicates the decreased glomerular filtration rate in abrin exposed groups. Clusterin
337
(Apolipoprotein J) is a 75 - 80 kDa disulfide-linked heterodimeric protein associated with the
338
clearance of cellular debris and apoptosis (Jones and Jomary, 2002). Down regulation of
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clusterin expression is positively correlated to cell apoptosis, and knockdown of clusterin
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expression results in an increase in proinflammatory cytokine-induced cell apoptosis. Clusterin
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expression was examined by Western blot and we found decreased expression of clusterin.
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Reduced clusterin expression results in an increase in cell death and renal tissue injury. These
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results are supported by histopathological findings in kidney. Our results indicate inflammatory
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changes in renal tubules, glomerular congestion and dilatation of proximal convoluted tubules
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besides infiltration of mononuclear cells. Our results confirm the anatomical and morphological
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changes in the kidney after abrin exposure. In an earlier study we reported dose and time
347
dependent neuroinflammatory damage mediated by infiltration of inflammatory cells into cortex
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region of brain (Bhasker et al., 2014). Abrin exposure resulted in the induction of rapid immune
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and inflammatory response in brain (Bhaskar et al., 2012). The present study is the first report on
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nephrotoxicity potential of abrin in mice.
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In conclusion, the study’s overall results signify abrin induced oxidative stress mediated
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nephrotoxicity in mice. The present study provides new insights into the nephrotoxic potential of
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abrin in terms of kidney oxidative stress, lipid peroxidation, and increased expression of TNF-α
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that induces the enhanced activity of MMP-9, ultimately leading to inflammation and kidney
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degeneration. Uremic toxins are capable of damaging other organs by crosstalk. Hence,
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protecting the kidneys during abrin poisoning is very essential for reducing death due to multi
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organ failure.
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Acknowledgements
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We thank Dr Lokendra Singh, Director, Defence Research and Development Establishment for
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providing all facilities and Dr Pravin Kumar, Head, Pharmacology and Toxicology Division for
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constant support during this study. Ms Bhavana is recipient of UGC Senior research fellowship.
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This work was supported by the grant from Ministry of Defence, India.
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Fig -1: Effect of 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal
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route on kidney GSH at 1, 3 and 7 days post-exposure. Values are mean ± SE of four mice per
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group.
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Fig-2: Effect of 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal
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route on kidney Lipid peroxidation at 1, 3 and 7 days post-exposure. Values are mean ± SE of
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four mice per group.
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Fig- 3(A): Immnoblot profile showing dose dependent and time course effect of 0.5 (0.88
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µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route on protein oxidation
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in kidney tissues.(B): Intensities of protein bands were quantified by densitometry. Results are
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expressed as histograms of kidney Oxy Blot, showing representative band pixel intensities
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(arbitrary units of densitometry analysis using NIH ImageJ software) and expressed as fold
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change in band pixel density over control. Data are presented as the mean ±SEM based of
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measurements done in tetraplicates. P<0.05 compared to control.
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Fig- 4(A): Immunoblot profile of TNF-α in kidney after 0.5 (0.88 µg/kg) and 1 LD50 (1.76
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µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure.(B): Intensities
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of protein bands were quantified by densitometric analysis. Data normalized to control.
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Fig- 5 (A): Gelatin zymogram of pro-MMP-9 and active MMP-9 after 0.5 (0.88 µg/kg) and 1
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LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure in
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kidney tissue.(B): Intensities of protein bands were quantified by densitometry. Data normalized
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to control. (C): Gelatin zymogram of serum MMP-9 and NGAL/MMP-9 complex after 0.5
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(0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7
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day post-exposure.(D, E): Densitometric analysis of serum NGAL/MMP-9 complex and serum
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MMP-9.
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Fig-6: Immunoblot of kidney injury biomarkers after 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg)
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of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure in kidney tissue. (A):
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expression of Cystatin-C. (B): expression of NGAL and (C): expression of Clusterin. (D): β-
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actin was used as protein loading control. The results shown are the representative of three
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separate experiments. Intensities of protein bands were quantified by densitometry. (E): Cystatin
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C, (F): NGAL, (G): Clusterin. Data normalized to control.
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Fig-7: Photomicrographs of control and abrin exposed mice kidney (H &E). (a): Control kidney
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section showing normal glomerulus, bowman’s space and renal parenchyma. (b): kidney section
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after 1 day post exposure with 0.5 LD50 abrin showing almost negligible degenerative changes.
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(c): kidney section after 3 days post exposure with 0.5 LD50 of abrin showing minimal to mild
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necrosis, atrophied glomeruli (arrow) and degenerative changes in tubular cells (arrow head).
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(d): kidney section 7 days post exposure with 0.5 LD50 abrin showing mesangial proliferations
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in glomeruli (arrow) degenerative changes in tubules (arrow head). (e): kidney section after 1
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day exposure with 1 LD50 abrin showing cellular necrosis (arrow) leading to degeneration of
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convoluted tubules. (f): kidney section after 3 days of exposure with 1 LD50 abrin showing
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extensive necrosed tubules (arrow) with vacuolation in glomeruli (arrow head) and degenerative
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changes in tubules, congestion in glomerulus and blood vessels.
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GSH
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Fig -1
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Protein Oxidation
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Kidney Zymography
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Serum Zymography
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Serum MMP-9/NGAL
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µmoles of GSH/gm of tissue
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Histopathology of kidney tissue
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HIGHLIGHTS Single exposure to abrin resulted in acute kidney injury.
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Abrin exposure in vivo results into oxidative stress mediated nephrotoxicity.
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Increased expression of TNF-α induces activity of MMP-9 which finally leads to inflammation and kidney damage
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Our results of kidney injury markers confirm the kidney function impairment.
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The animals were handled according to the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals). The experiment was approved by Institutional Animal Ethics Committee, a statutory committee constituted by
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CPCSEA, Animal Welfare Cell, Ministry of Environment, Forests and Climate Change,
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Government of India.
PII:
S0041-0101(17)30094-6
DOI:
10.1016/j.toxicon.2017.03.007
Reference:
TOXCON 5594
To appear in:
Toxicon
Received Date: 25 August 2016 Revised Date:
8 March 2017
Accepted Date: 9 March 2017
Please cite this article as: Sant, B., Rao, P.V.L., Nagar, D.P., Pant, S.C., Bhasker, A.S.B., Evaluation of abrin induced nephrotoxicity by using novel renal injury markers, Toxicon (2017), doi: 10.1016/ j.toxicon.2017.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of abrin induced nephrotoxicity by using novel renal injury markers
2 Bhavana Sant, P.V. Lakshmana Rao, D.P. Nagar, S.C. Pant and A. S. B. Bhasker*
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Division of Pharmacology and Toxicology, Defence Research and Development Establishment,
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Jhansi Road, Gwalior 474002, India.
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*Corresponding Author:
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Tel: +91-751-2233495 Fax: +91-751-2341148
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E-mail: [email protected]
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Abstract
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Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus
24
precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes
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cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study
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we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress,
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inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed
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to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-
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toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were
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observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as
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observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by
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using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney
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injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity
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of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due
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to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of
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abrin.
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1. INTRODUCTION
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Abrin and ricin are among the most toxic plant proteins (Roxas-Duncan and Smith, 2012). Both
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proteins are derived from the seeds of the plants Abrus precatorius and Ricinus communis
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respectively. Abrin shows significant similarities to ricin both at the sequence and structural
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levels; however abrin is a more potent toxin than ricin (Surendranath and Karande, 2008). Both
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toxins belong to the family of type II Ribosome Inactivating Proteins (RIPs). Toxins from the
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RIP family irreversibly inactivate protein synthesis through an enzymatic mechanism. The
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reported human fatal dose of abrin is 0.1-1µg/kg of body weight, with a molecular mass of
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approximately 60-65KDa (Wooten et al., 2014). Abrin is a protein synthesis inhibitor and
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induces apoptosis in cells (Mishra and Karande, 2014). Abrin is a heterodimer consisting of two
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disulfide-linked polypeptides, known as the A-chain and B-chain. The active A-chain moiety has
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an enzymatic function. The toxicity of A-chain is due to its RNA N-glycosidase activity by
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which it brings about depurination of adenine at 4324 position in the 28S ribosomal RNA. The
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end result of this activity is complete inhibition of protein synthesis leading to cell death (Olsnes,
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2004). The B chain has galactose-binding capability and shows lectin type activity, helping the
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toxin to internalize inside the cell.
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subsequently at higher doses, severe necrosis in the organs of poisoned animals and in cultured
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cells (Griffiths et al., 1987; Narayanan et al., 2004). Earlier, we investigated oxidative stress
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mediated DNA damage and cell death by using ricin and abrin in vitro (Rao et al., 2005; Bhaskar
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et al., 2008).
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Accidental or intentional exposure to abrin has been frequently reported (Sahni et al., 2007;
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Sahoo et al., 2008). Many of these poisoning cases showed unusual symptoms like central
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nervous system toxicity and renal failure (Subramanyam, 2008).
At cellular level both abrin and ricin cause apoptosis and
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Nephrotoxicity is an important target of toxicological studies due to its crucial role in drug
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excretion and detoxification. A number of studies have described neuroinflammation and
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damage caused by nephrotoxicity (Burn and Bates, 1998; Liu et al., 2008; Brouns and Deyn,
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2004). Various nephrotoxicants produce different types of toxicity syndromes, including acute
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renal failure, chronic renal failure, renal tubular defects, nephrotic syndromes and hypertension.
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Tubular kidney cells are particularly vulnerable to toxin mediated acute kidney injury due to
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their disproportionate exposure to circulating chemicals and transport processes that result in
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high intracellular concentration. Due to abundant presence of polyunsaturated fatty acids in the
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composition of renal lipids, kidneys are very susceptible to oxidative stress (Ozbek, 2012). RIPs
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like trichosanthin and shiga toxin have been reported to cause kidney injury via tubular damage
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(Tang et al., 1997; Porubsky et al., 2014). Ricin induced nephrotoxicity is characterized by
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altered serum biomarkers of kidney function, increased lipid peroxidation and decreased activity
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of antioxidant enzymes, leading to oxidative stress induced renal toxicity (Kumar et al., 2003).
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Oxidative stress, which occurs when there are excessive free radical production or low
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antioxidant levels, has been reported in chronic kidney disease conditions. These free radicals
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can damage proteins, lipids, carbohydrates and nucleic acids. Oxidative stress leads to tissue
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damage through different mechanisms, including lipid peroxidation, DNA damage and protein
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modification. These mechanisms have been implicated in the pathogenesis of several systemic
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diseases including kidney disease (Ozbek, 2012). Glutathione serves to protect cells from
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oxidative stress. Glutathione, normally present in high amounts in tubular cells, can neutralize
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ROS. Reduced kidney cellular glutathione levels and depletion of other antioxidants lead to
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oxidative stress in cells (Abraham et al., 2013). Inflammation is now believed to play a major
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role in the pathophysiology of acute kidney injury. Leukocytes and renal tubular cells release
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many cytokines into the injured kidney and are important components of both the initiation and
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extension of inflammation during acute kidney injury (Akcayet et al., 2009). TNF-α mediated
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kidney injury has also been reported to follow exposure to shiga toxin, a known RIP of bacterial
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origin (Lentz et al., 2010).
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Oxidative stress may increase the production of TNF-α and other cytokines by activation
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of NFκB (Manna et al., 1998). TNF-α is a proinflammatory marker produced by immune cells
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like macrophages and lymphocytes; however, further studies revealed that it is also produced by
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endothelial and epithelial cells (Ramseyer and Garvin, 2013). Recruitment of immune cells into
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the kidney, which releases cytokines, may cause inflammation and cell death (Zhu et al., 2009).
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TNF-α is a strong inducer of matrix metalloproteinase, MMP-9 (Baharet et al., 2010). Zhou et al.
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(2009) described TNF-α induced expression of MMP-9 mediated by activation of p21-activated-
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kinase-1 and JNK pathway. Matrix metalloproteinases (MMPs) are enzymes responsible for
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degradation of the extracellular matrix (ECM) and are involved in the pathogenesis of ischemia-
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re-perfusion injury. MMP‑9 is gelatinase and cleaves to denatured collagens (gelatins) and
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laminin, as well as certain chemokines. In the kidney, MMP’s are synthesized by intrinsic
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glomerular cells and tubular epithelial cells. ECM degrading ability of MMPs play an important
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role in the progression of nephropathies. Altered MMP expression, like ability of MMP-2 to
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induce or sustain an inflammatory mesangial cell phenotype, has been observed in a large
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number of kidney diseases (Marti, 2000).
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Some of the prominent biomarkers of kidney injury include NGAL (Neutrophil Gelatinase
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Associated Lipocalin), Cystatin C and Clusterin. (Fassett et al., 2011). NGAL, a ubiquitous 25
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KDa protein, covalently bound to gelatinase from human neutrophils, is a marker of tubular
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injury (Cowland and Borregaard, 1997; Dharnidharka et al., 2002). Serum and urine NGAL
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levels predict acute kidney injury in different clinical conditions (Mishra et al., 2005). Cystatin C
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is produced in all nucleated cells. This protein is a marker of kidney injury from drug toxicity
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and many chronic diseases. It is a marker of glomerular filtration rate and also indicates proximal
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tubule injury. Clusterin is a multifunctional glycoprotein with roles in metabolism and transport
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of lipids. Reduced clusterin expression results in an increase in cell death or renal tissue injury.
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The mechanism behind clusterin’s kidney protection is unclear. Up regulation of clusterin
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expression may cause anti-apoptosis and inactivation of complements which may partly
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contribute in protection of kidney injury (Zhou et al., 2010).
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The renal response to poisons is dynamic, and the kidney adapts to maintain homeostasis during
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the cascade of repair and recovery that follows the primary insult (Bach,1989). There are reports
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on abrin-induced neurotoxicity (Bhasker et al., 2014) and hepatotoxicity, but little information is
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available about the nephrotoxicity potential of abrin. Hence, the objectives of this study were to
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determine the following:
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a) The mechanism of acute kidney injury after abrin exposure in a mouse model by using
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different kidney specific markers and histopathology.
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b) The role of inflammation, proximal tubular damage, oxidative stress, and vascular injury on
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the pathogenesis of abrin-induced nephrotoxicity.
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2. MATERIALS AND METHODS
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2.1 Animals
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The study used BALB/c mice randomly bred in the Institute’s animal facility, weighing between
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23 and 27 g. The animals were maintained according to standard conditions of temperature and
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humidity (25±20C, relative humidity 40-60%), and the animals were fed a standard pellet diet
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(Ashirwad Brand, Chandigarh, India). Food and water were given ad libitum. The animals were
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handled according to the guidelines of CPCSEA (Committee for the Purpose of Control and
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Supervision on Experiments on Animals). The study was approved by Institutional Animal
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Ethics Committee, a statutory committee constituted by CPCSEA, Animal Welfare Cell,
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Ministry of Environment, Forests and Climate Change, Government of India.
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2.2 Chemicals
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Abrin was isolated from A. precatorius seeds as described elsewhere (Kumar et al., 2008). The
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purified abrin was lyophilized and stored at −80 ◦C and reconstituted as and when required by
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PBS. Reduced glutathione (GSH) was from Across (Belgium). O-phthaldialdehyde (OPT) was
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from Fluka (USA). All other chemicals were of extra pure grade and obtained from Sigma
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Chemical Co. (St. Louis, USA) unless otherwise mentioned.
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2.3 Experimental design
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The mice were divided into six groups: Control, 0.5LD50 1, 3 and 7 day, and 1LD50 1 and 3
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day. Because not all animals survived to 7 days in the 1LD50 dose group, data for 7 day is not
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included in the data analysis. All treated groups including control consisted of six animals each.
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Four animals were used for biochemical assays and two for performing histopathology. 0.5LD50
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(0.88µg/kg) and 1LD50 (1.76µg/kg) dose of abrin toxin was administered intraperitoneally in
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mice. Control mice received an equal volume PBS by same route. Mice were handled in
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accordance with the standard guide for the care and use of laboratory animals. At the end of
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experimental period, animals were sacrificed by cervical dislocation under deep anesthesia.
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Following induction of anesthesia, the mice were subjected to a midline abdominal incision and
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both left and right kidney tissues were harvested.
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2.4 Biochemical assays
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GSH level was measured spectrofluorimetrically using the method of Hissin and Hilf (1976) and
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expressed as µmloes of GSH/ g tissue. Malondialdehyde (MDA) content was evaluated using the
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thiobarbituric acid (TBA) (Ohkawa et al., 1979). Absorbance was measured at 532 nm to
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determine the MDA content. The level of lipid peroxidation in kidneys is expressed as nmoles of
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MDA/g tissue.
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2.5 Western blot analysis of TNF-α, Clusterin, Cystatin C, NGAL.
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Polyclonal anti-TNF-α, anti-Clusterin, and anti-NGAL antibodies were purchased from Santa
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Cruz Biotechnology Inc. Monoclonal anti- Cystatin C was purchased from Abcam (UK). Total
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protein was estimated using a Bio-Rad kit, separated by SDS‑PAGE and transferred to
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nitrocellulose membranes. The membrane was treated with a blocking solution (5% skimmed
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milk in TBST) and incubated overnight at 4°C in shaking condition, with various primary
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antibodies at specified dilutions TNF-α (1:500), Clusterin (1:500), NGAL (1: 1000), Cystatin C
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(1:10,000). The membranes were washed four times in TBST for 7 minutes each, followed by
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incubation for 2 hours in horseradish peroxidase-conjugated anti-rabbit and anti-goat secondary
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antibody (1:20000). Membranes were washed again and immunoreactive proteins were
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visualized
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(ProteoQwestTMChemiluminescent Western blotting kit, Sigma-Aldrich), and images were taken
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on a Fusion FX5 image analyser. After western blot analysis, each band was analyzed using
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using
an
enhanced
chemiluminescence
detection
kit
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ImageJ software for quantitative measurement. In short, a constant unit area was selected and
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average density of the total band area of the respective proteins in control and treated groups
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were measured and represented graphically.
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2.6 Protein carbonyl assay
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Carbonylated proteins of kidney tissues were detected using Oxyblot™ Protein Oxidation
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Detection Kit (Chemicon/Millipore). Briefly, the protein samples were prepared and carbonyl
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groups were derivatized using 1x DNPH solution provided in the kit, then the samples were
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resolved on SDS-PAGE, transferred on to nitrocellulose membrane. DNP-derivatized proteins
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were identified by chemiluminescent method using an anti-DNP antibody. Carbonylated protein
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bands were quantified by densitometry using NIH ImageJ software.
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2.7 Substrate gel zymography
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The extracellular activities of MMP-9 in tissue and serum were assessed using gelatin
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zymography. Tissue was homogenized in a lysis buffer containing protease inhibitors. The
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homogenate was centrifuged for 30 min at 10,000x rpm, and supernatant was used as protein
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lysate. Protein content was estimated using Bio-Rad kit, and 40µg of protein was resolved in 8%
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non-reducing SDS electrophoresis gel containing 0.4% gelatin. The gel was washed with
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washing buffer (50 mM Tris–HCl pH 7.5, 2.5% Triton X-100) for 2 h at room temperature and
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incubated overnight in developing buffer (50 mM Tris–HCl pH 7.5, 10 mM CaCl2, 1 M ZnCl2)
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at 370C. Protein bands were visualized using Coomassie blue staining followed by destaining.
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Proteolytic activities were quantified by performing densitometry using NIH ImageJ software
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and results presented graphically.
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2.8 Histopathological examination
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Control and abrin treated animals were sacrificed after specific time points. Tissue samples of
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kidneys were dissected. After fixation small pieces were processed by automated tissue
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processor (Leica TP 1020), dehydrated and embedded in paraffin wax. Multiple sections of 4-5
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µm thick from each block were cut on a rotatory microtome (Microm, Germany), mounted on a
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slide with albumin coating and air dried overnight. The sections were stained with hematoxylin
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and eosin (McMannus and Mowry, 1965) in Leica Autostain-XL. Microscope observation was
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performed under Leica, DMLB microscope and photographs were taken using a DM 500 camera
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(Leica, Germany).
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2.9 Statistical analysis
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One-way ANOVA followed by Dunnet’s test were performed and used for comparison between
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the control and treated groups. The level of significance was set at P ≤ 0.05.
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3. RESULTS
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3.1 Effect of abrin on oxidative stress parameters
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Reduced glutathione, an important intracellular antioxidant, decreased in the kidneys of mice
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treated with 0.5 and 1 LD50 doses of abrin at different time points (Fig. 1). Time dependent
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decrease was found in the 1 LD50 group, and although this pattern was not observed in 0.5LD50
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group, the decrease was still significantly different from the control. On day 1 the GSH levels
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were 1.79 ±0.11, and 1.58 ± 0.20 on day 3. On day 7 GSH levels were 1.77 ± 0.27. Control
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levels of GSH were 3.44 ±0.23. As compared to 0.5LD50 dose, GSH levels were more reduced
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on day 1 and on day 3 of 1LD50 dose (1.37 ± 0.17 & 1.33 ± 0.17). GSH concentration was
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measured in µmoles of GSH/gm of tissue.
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Time dependent increase in lipid peroxidation was observed in the 1LD50, and not in the
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0.5LD50 group (Fig. 2). But MDA concentration in 0.5LD50 group was significantly higher than
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control.
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3.2 Effect on oxidation of kidney proteins and TNF-α
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Protein carbonyl content is not only a biomarker of oxidative stress, but also provides evidence
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of oxidative protein damage. An increased number of oxidized protein bands, which are more
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intense in levels, were detected in a dose and time dependent manner in all the treated groups at
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all the time points compared to control (Fig. 3A-B). Western blot assessment of TNF-α was
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carried out. Time dependent increase in expression of TNF-α was found in 0.5LD50 group as
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compared to control. This increase was also observed in 1LD50 but was not time dependent (Fig.
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4A-B).
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3.3 Kidney and serum zymography
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Zymographic analysis of kidney tissue revealed gelatinolytic activity of pro-MMP-9 and active
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MMP-9 (92 KDa ). Differences were found in the activities of both gelatinolytes as compared to
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control. Increased activity of MMP-9 was observed in abrin treated group (Fig. 5A-B). Serum
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levels of MMP-9 were also analyzed by zymography. At 1LD50 there was time dependent
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increase in MMP-9. A high-molecular-weight complex of NGAL/MMP-9 (125 KDa) was also
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observed in serum. Higher activity of this complex was found at both doses and time points in
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the abrin exposed group compared to control (Fig. 5C). Quantitative analysis through ImageJ
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software is shown in Fig. 5 D-E.
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3.4 Western blot analysis of kidney injury biomarkers
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Neutrophil gelatinase-associated lipocalin (NGAL, also called lipocalin 2 or 24p3), a small 25
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kDa protein that belongs to lopocalin family, is highly expressed during acute renal injury in
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animal models. Protein expression of NGAL was increased in dose and time dependent manner.
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Clusterin is a lipoprotein that has anti-complement effects in membranous nephropathy. Time
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dependent decrease in expression of clusterin was found in the 0.5LD50 group as compared to
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the control. In the 1LD50 group, clusterin decreased more by day 3 than day 1, indicating the
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severity of tubular damage.
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Cystatin C, cysteine protease inhibitor, is a marker for decreased glomerular filtration rate.
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Immunoblot results have shown increased expression of cystatin C in all treated groups as
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compared to the control. Time dependent increase was found in the 0.5LD50 group, but this
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pattern was not seen in the 1LD50 group. No expression was observed in the control group
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(Fig.6A-D). Quantitative analysis of the blots through ImageJ software revealed that the changes
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were significantly different from control (Fig.6E-G).
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3.5 Kidney histology
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The kidney histopathology of mice exposed to abrin sacrificed on days 1, 3 and 7 is shown in
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Fig.7 (a-f). The kidneys of control mice showed normal architecture (Fig. 7a), including normal
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glomerulus, Bowman's space and renal parenchyma. One day after exposure to 0.5 LD50 abrin
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showed negligible degenerative changes. Moderate changes appeared after 3 and 7 days in the
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kidneys of mice treated with same dose, which shows that accumulation of edematous fluid in
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the medullary areas is associated with infiltration of inflammatory cells (Fig. 7c-d). The kidneys
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of mice one day after exposure to1 LD50 abrin showed cloudy swelling in the renal tubule with
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vacuolar degeneration (Fig. 7e). By day 3 they showed severe hemorrhage, glomerular
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congestion, dilatation of the proximal convoluted tubules, obliteration of renal parenchyma, and
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loss of epithelial lining, and the lumen of these tubules was filled with eosinophilic material and
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cell exudate. These changes were associated with infiltration of mononuclear cells (Fig. 7f).
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4. DISCUSSION
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Ribosome inactivating proteins are a group of proteins that share the property of damaging
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ribosomes in an irreversible manner acting catalytically. One of the most potent members of the
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class II RIP family is the phytotoxin abrin produced by the subtropical climber Abrus
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precatorius. Abrin inhibits elongation factor EF-1 and EF-2, preventing protein synthesis and
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leading to cell death (Bradberry, 2004; Robertus, 1991). Due to its high availability and ease of
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preparation, abrin is considered a biological threat, especially in context of bioterrorism. To date,
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there is no established therapeutic countermeasure against abrin intoxication.
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There are some reported cases of abrin poisoning in the USA and India (Roxas-Duncan and
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Smith, 2012), and cases include both accidental and intentional ingestion of seeds. Children are
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at greater risk of ingesting seeds due to their colorful appearance, and the consequences could be
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fatal. After consuming abrus seeds, an 18-month-old child reported with gastrointestinal
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symptoms which were consistent with reported cases of abrin poisoning, but the patient also had
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isolated and significantly elevated alkaline phosphatase levels. Tests could not reveal the
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responsible pathological process (Mazin et al., 2015).
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Abrin is toxic by all routes of exposure, although its lethality depends on its route of
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administration (inhalation, ingestion, or injection) (Goldman et al., 2011). Our laboratory
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recently reported neurodegenerative changes after intraperitoneal abrin exposure in mice
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(Bhasker et al., 2014). Nephrotoxicity of abrin has not been reported before, although Wooten et
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al. (2014) presented a case of abrin poisoning of a 22-month-old girl, who developed a
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remarkable increase in serum kidney markers like creatinine and BUN. In a case report of abrin
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poisoning, a 17-year-old woman showed signs of CNS toxicity and altered sensorium. The
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patient also developed renal failure and an enlarged kidney, hyponatremia (Subrahmanyan et al.,
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2008). Based on above data on abrin poisoning, the present study was designed to evaluate the
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nephrotoxic potential of abrin and possible mechanism mediating nephrotoxicity.
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Reports are available on direct renal injury against other ribosome-inhibiting proteins like shiga
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toxin, which induces apoptosis and TNF-gene expression in the medulla, monocyte/macrophage
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infiltration, platelet aggregation, severe tubular damage and acute deterioration of renal function
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(Yamamoto et al., 2005). Renal impairment reported after infusion of trichosanthin, a type I
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ribosome inactivating protein, resulting in reduction of glomerular filtration rate and tubular
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proteinuria. Both necrotic cell death and apoptosis participated in cell loss from the proximal
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tubules. Such toxicity may be mediated through intracellular events induced by trichosanthin
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(Tang et al., 1997). A report for ricin toxicity has revealed that ricin administered with or without
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lipopolysacharide rapidly produced oliguric renal failure, hemolysis and thrombocytopenia.
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Inflammatory response was also seen with a marked increase in plasma concentrations of
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inflammatory cytokines and a glomerular infiltration of macrophages (Taylor, 1999).
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Several lines of evidence support the hypothesis that ROS are involved in the pathophysiology of
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renal failure (Nath and Norby, 2000). Oxidative stress is seen both systemically and locally at the
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kidney level through the enhancement of ROS production by circulating neutrophils and
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membrane lipid peroxidation in kidney respectively. Carbonylated proteins of abrin exposed
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kidney tissue were detected using OxyBlotTM. Significant increase in oxidized proteins with dose
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and time dependent changes confirms protein oxidation after abrin exposure. Reduction of GSH
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in the kidney contributes to renal epithelial cell death (Gomez et al., 2013). Taken together, our
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results of depleted GSH concentrations and increased MDA content suggests that abrin induces
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oxidative stress and renal epithelial cell damage. Inflammation is one of the manifestations of
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oxidative stress, and the pathways that generate the mediators of inflammation, such as adhesion
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molecules and interleukins, are all induced by oxidative stress.
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Tumor necrosis factor (TNF) alpha is a potent pro-inflammatory cytokine and important
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mediator of inflammatory tissue damage. There are number of reports on TNF-α induced renal
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damage. It may be due in part to recruitment of immune cells into the kidney, releasing cytokines
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that cause inflammation and cell death (Zhu et al., 2009). TNF-α may contribute to abrin
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nephrotoxicity by inciting an inflammatory response within the kidney. It is also likely due to
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direct actions of TNF-α on renal cells. Increased expression and activity of TNF- α after abrin
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exposure directly indicates kidney inflammation. There are several reports detailing TNF-α
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induced expression of matrix metalloproteinases (MMPs). MMPs regulated by various stimuli
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are important in remodeling glomerular ECM, which leads to a number of renal diseases. TNF-α
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was involved in the regulation of MMPs and signal pathways involved in TNF-α-induced MMPs
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expression in rat glomerular mesangial cells (Wang et al., 2014). Expression of MMP-2 and
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MMP-9 has been detected in glomerulus and proximal tubules of rats (Catania et al., 2007).
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There are several reports that support the idea that MMPs mediate acute kidney injury and are
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involved in changes in the glomeruli and tubular epithelial cells (Romero et al., 2009). In the
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present study, our results show TNF-α increased activity of kidney tissue pro-MMP-9 and active
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MMP-9 in the abrin treated group, leading to inflammatory changes. Pro-MMP-9 can also be
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activated in various diseased conditions such as ischemia, a hallmark of acute renal failure. It
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strongly stimulated proteolytic activity of proMMP-9 and MMP-9 in the renal endothelial
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fractions (Caron et al., 2005). NGAL is a biomarker of tubular injury. NGAL protects MMP-9
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from proteolytic degradation and enhances its enzymatic activities by binding and forming the
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MMP-9/NGAL complex. Therefore, NGAL, MMP-9 and their complex MMP-9/NGAL have
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been proposed as biomarkers for numerous malignancies (Carlo, 2013). We found increased
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expression of NGAL/MMP-9 complex in sera of the abrin treated group. Recent data
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demonstrated that increased levels of NGAL are present in chronic kidney disease and acute
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kidney damage (Chakraborty et al., 2012; Bolignano et al., 2008).
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Further evidence for abrin induced kidney damage in the present study was obtained by studying
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other protein markers in the kidney – namely cystatin-C and clusterin – that together offer
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information on location of injury in the nephron. Cystatin C is a biomarker of the glomerular
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filtration rate (Cabarkapa, 2015) that is produced in the nucleated cells at a constant amount, and
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its serum concentration does not depend on muscle mass or protein intake. The catabolism of
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cystatin C is mostly done in the kidneys. Our results have shown increased expression of cystatin
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C which indicates the decreased glomerular filtration rate in abrin exposed groups. Clusterin
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(Apolipoprotein J) is a 75 - 80 kDa disulfide-linked heterodimeric protein associated with the
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clearance of cellular debris and apoptosis (Jones and Jomary, 2002). Down regulation of
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clusterin expression is positively correlated to cell apoptosis, and knockdown of clusterin
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expression results in an increase in proinflammatory cytokine-induced cell apoptosis. Clusterin
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expression was examined by Western blot and we found decreased expression of clusterin.
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Reduced clusterin expression results in an increase in cell death and renal tissue injury. These
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results are supported by histopathological findings in kidney. Our results indicate inflammatory
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changes in renal tubules, glomerular congestion and dilatation of proximal convoluted tubules
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besides infiltration of mononuclear cells. Our results confirm the anatomical and morphological
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changes in the kidney after abrin exposure. In an earlier study we reported dose and time
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dependent neuroinflammatory damage mediated by infiltration of inflammatory cells into cortex
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region of brain (Bhasker et al., 2014). Abrin exposure resulted in the induction of rapid immune
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and inflammatory response in brain (Bhaskar et al., 2012). The present study is the first report on
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nephrotoxicity potential of abrin in mice.
351
In conclusion, the study’s overall results signify abrin induced oxidative stress mediated
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nephrotoxicity in mice. The present study provides new insights into the nephrotoxic potential of
353
abrin in terms of kidney oxidative stress, lipid peroxidation, and increased expression of TNF-α
354
that induces the enhanced activity of MMP-9, ultimately leading to inflammation and kidney
355
degeneration. Uremic toxins are capable of damaging other organs by crosstalk. Hence,
356
protecting the kidneys during abrin poisoning is very essential for reducing death due to multi
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organ failure.
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Acknowledgements
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We thank Dr Lokendra Singh, Director, Defence Research and Development Establishment for
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providing all facilities and Dr Pravin Kumar, Head, Pharmacology and Toxicology Division for
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constant support during this study. Ms Bhavana is recipient of UGC Senior research fellowship.
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This work was supported by the grant from Ministry of Defence, India.
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Fig -1: Effect of 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal
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route on kidney GSH at 1, 3 and 7 days post-exposure. Values are mean ± SE of four mice per
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group.
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Fig-2: Effect of 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal
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route on kidney Lipid peroxidation at 1, 3 and 7 days post-exposure. Values are mean ± SE of
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four mice per group.
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Fig- 3(A): Immnoblot profile showing dose dependent and time course effect of 0.5 (0.88
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µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route on protein oxidation
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in kidney tissues.(B): Intensities of protein bands were quantified by densitometry. Results are
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expressed as histograms of kidney Oxy Blot, showing representative band pixel intensities
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(arbitrary units of densitometry analysis using NIH ImageJ software) and expressed as fold
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change in band pixel density over control. Data are presented as the mean ±SEM based of
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measurements done in tetraplicates. P<0.05 compared to control.
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Fig- 4(A): Immunoblot profile of TNF-α in kidney after 0.5 (0.88 µg/kg) and 1 LD50 (1.76
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µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure.(B): Intensities
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of protein bands were quantified by densitometric analysis. Data normalized to control.
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Fig- 5 (A): Gelatin zymogram of pro-MMP-9 and active MMP-9 after 0.5 (0.88 µg/kg) and 1
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LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure in
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kidney tissue.(B): Intensities of protein bands were quantified by densitometry. Data normalized
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to control. (C): Gelatin zymogram of serum MMP-9 and NGAL/MMP-9 complex after 0.5
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(0.88 µg/kg) and 1 LD50 (1.76 µg/kg) of abrin exposure by intraperitoneal route at 1, 3 and 7
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day post-exposure.(D, E): Densitometric analysis of serum NGAL/MMP-9 complex and serum
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MMP-9.
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Fig-6: Immunoblot of kidney injury biomarkers after 0.5 (0.88 µg/kg) and 1 LD50 (1.76 µg/kg)
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of abrin exposure by intraperitoneal route at 1, 3 and 7 day post-exposure in kidney tissue. (A):
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expression of Cystatin-C. (B): expression of NGAL and (C): expression of Clusterin. (D): β-
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actin was used as protein loading control. The results shown are the representative of three
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separate experiments. Intensities of protein bands were quantified by densitometry. (E): Cystatin
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C, (F): NGAL, (G): Clusterin. Data normalized to control.
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Fig-7: Photomicrographs of control and abrin exposed mice kidney (H &E). (a): Control kidney
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section showing normal glomerulus, bowman’s space and renal parenchyma. (b): kidney section
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after 1 day post exposure with 0.5 LD50 abrin showing almost negligible degenerative changes.
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(c): kidney section after 3 days post exposure with 0.5 LD50 of abrin showing minimal to mild
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necrosis, atrophied glomeruli (arrow) and degenerative changes in tubular cells (arrow head).
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(d): kidney section 7 days post exposure with 0.5 LD50 abrin showing mesangial proliferations
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in glomeruli (arrow) degenerative changes in tubules (arrow head). (e): kidney section after 1
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day exposure with 1 LD50 abrin showing cellular necrosis (arrow) leading to degeneration of
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convoluted tubules. (f): kidney section after 3 days of exposure with 1 LD50 abrin showing
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extensive necrosed tubules (arrow) with vacuolation in glomeruli (arrow head) and degenerative
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changes in tubules, congestion in glomerulus and blood vessels.
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GSH
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Fig -1
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Protein Oxidation
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Fig – 3 (A)
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µmoles of GSH/gm of tissue
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Kidney Zymography
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Serum Zymography
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Serum MMP-9/NGAL
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Fig - 5 (D)
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Serum MMP-9
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*
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µmoles of GSH/gm of tissue
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Fig – 5 (E)
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0.5LD50
C
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CYSTATIN – C 15KDa
Fig – 6 (A)
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0.5LD50
C
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1D
Fig – 6 (C)
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*
669 670 671
150
*
*
*
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Fig – 6 (E)
Control
1 Day
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7 Day
672
µmoles of GSH/gm of tissue
674 675
Fig – 6 (F)
683
*
*
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0
Control
1 Day
3 Day
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0.5 LD50
3 Day 1 LD50
Fig – 6 (G)
Clusterin 140
*
120 µmoles of GSH/gm of tissue
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*
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µmoles of GSH/gm of tissue
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* 100
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Histopathology of kidney tissue
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HIGHLIGHTS Single exposure to abrin resulted in acute kidney injury.
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Abrin exposure in vivo results into oxidative stress mediated nephrotoxicity.
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Increased expression of TNF-α induces activity of MMP-9 which finally leads to inflammation and kidney damage
•
Our results of kidney injury markers confirm the kidney function impairment.
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•
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The animals were handled according to the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals). The experiment was approved by Institutional Animal Ethics Committee, a statutory committee constituted by
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CPCSEA, Animal Welfare Cell, Ministry of Environment, Forests and Climate Change,
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Government of India.