Modulation of lithiatic injury to renal epithelial cells by aqueous extract of Terminalia arjuna

Accepted Manuscript Title: Modulation of lithiatic injury to renal epithelial cells by aqueous extract of Terminalia arjuna Authors: Amisha Mittal, Simran Tandon, Surender Kumar Singla, Chanderdeep Tandon PII: DOI: Reference:

S2210-8033(18)30003-4 https://doi.org/10.1016/j.hermed.2018.01.003 HERMED 207

To appear in: Received date: Revised date: Accepted date:

14-12-2016 17-11-2017 7-1-2018

Please cite this article as: Mittal, Amisha, Tandon, Simran, Singla, Surender Kumar, Tandon, Chanderdeep, Modulation of lithiatic injury to renal epithelial cells by aqueous extract of Terminalia arjuna.Journal of Herbal Medicine https://doi.org/10.1016/j.hermed.2018.01.003 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.

Modulation of lithiatic injury to renal epithelial cells by aqueous extract of Terminalia arjuna Authors: Amisha Mittala, Simran Tandonb, Surender Kumar Singlac, Chanderdeep Tandond

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Institution: a Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan - 173234, Himachal Pradesh, India b Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University Uttar Pradesh, Noida - 201313, U.P., India c Department of Biochemistry, Panjab University, Chandigarh - 160014, India d Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector - 125, Noida - 201313, U.P., India

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Corresponding author: Prof. (Dr.) Chanderdeep Tandon Director, Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector - 125, Noida - 201313, U.P., India Telephone No.: +91(0) -120-439 -2195, +919871003672 Fax: +91(0) -120-439 -2947 E-mail: [email protected]

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Graphical abstract

Highlights  This study is aimed to establish a scientific basis for the anti-urolithiatic property of Terminalia arjuna.

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The aqueous extract of T. arjuna bark was evaluated against oxalate induced injury to renal tubular epithelial cells using in vitro model of NRK52E. The aqueous extract significantly improved the cell viability in a concentration dependent manner. The aqueous extract effectively attenuated the retention of CaOx crystals to apical surface of renal cells. The aqueous extract substantially diminished oxalate induced apoptotic death of renal cells.

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Abstract: Previous studies have shown that hyperoxaluria is concomitant with the formation of CaOx crystals and the subsequent propensity of these crystals towards renal cells greatly increases the risk for the development of urolithiasis. Despite advances in surgical management, recurrence of stones and side effects of present day treatment persists and in the light of this a cost-effective substitute from natural sources such as phytotherapy is being sought. The present study was designed to investigate the antiurolithiatic efficacy of a single plant preparation comprising of an aqueous extract (AE) of the bark of Terminalia arjuna (T. arjuna) on oxalate injured cells. We used an in vitro model system comprising of a normal epithelial cell line (NRK-52E) which was exposed to 2 mM oxalate for 48 hours, following which the cytoprotective potential of AE on cell viability, CaOx crystal adherence and apoptotic changes were evaluated. The results revealed that co-treatment with T. arjuna AE to cells exposed to 2 mM oxalate for 48 hours, rendered protection from oxalate triggered damage. On treatment with different concentrations of the T. arjuna AE, the cell viability increased in a concentration dependent manner. Moreover, the extract prevented the interaction of the CaOx crystals to the cell surface and reduced the number of apoptotic cells. The current data suggests that T. arjuna bark aqueous extract could be a potential phytotherapeutic treatment against urolithiasis based on its ability to diminish oxalate induced morphological changes, apoptosis and death of renal cells thereby leading to cell survival.

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Abbreviations CaOx: Calcium oxalate AE: Aqueous extract T. arjuna: Terminalia arjuna NRK-52E: Normal renal tubular epithelial cell line COM: Calcium oxalate monohydrate COD: Calcium oxalate dihydrate DMSO: Dimethyl sulfoxide DMEM: Dulbecco’s Modified Eagles’s Medium NCCS: National Centre for Cell Science MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PBS: Phosphate buffered saline PS: Phosphatidylserine PI: Propidium iodide

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Key words: Urolithiasis; Calcium oxalate; Renal epithelial cells; Normal Rat Kidney Epithelial Cells; Phytotherapy; Terminalia arjuna

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Introduction

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Urolithiasis is the third most common multifactorial urological condition (Fisang et al., 2015), affecting around 12% of the population worldwide (Joy et al., 2012), however, the exact mechanism behind the origin of kidney stones is still obscure. CaOx stones account for the majority of kidney stones formed (Menon and Koul, 1992) and appear in two distinct morphological forms, calcium oxalate monohydrate (COM) or Whewellite, and calcium oxalate dihydrate (COD) or Weddellite, which exhibit different pathologies. COM is more thermodynamically stable than COD and has greater affinity for renal tubular epithelial cells (Verkoelen et al., 1995). Under pathophysiological condition, they adhere to the apical surface of renal cells rapidly (Riese et al., 1988 and Lieske et al., 1996) followed by internalization (Lieske et al., 1994, 1992). Once inside a cell, they are either digested by lysosomes (Lieske et al., 1997) or initiate a cascade of signals, consequently, leading to a CaOx stone formation (Khan, 1997 and Jonassen et al., 2003). The formation of CaOx crystals is a natural consequence of the electrostatic interaction of oxalate ions with the calcium ions which are present in high concentrations in the renal tubular fluid (Lieske et al., 1994) of both nonstone and stone formers. Normally these crystals are freely excreted into the urine (Finlayson et al., 1984)

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however, under certain pathological scenarios these crystals develop into stones and are retained within the kidney. Subsequently, they cause injury to tubular cells and present a site for the further formation of a stone nidus, which overtime enhances in size by subsequent ion or crystal deposition (Fasano and Khan, 2001). Crystal–cell interaction is also stimulated by renal tubular damage and thus, retention of these crystals on cell surface is a key step linking extracellular and intracellular events (Khan, 2004).

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Although major advances have been seen for the management of kidney stones they do not address the issue of recurrence which is responsible for the significant trauma to these patients with renal calcifications however, which if not controlled can ramify into the eventual destruction of the kidney (Verkoelen, 2006). To overcome the tenacious side effects of the current treatments (surgery, extracorporeal shockwave lithotripsy, percutaneous nephrolithotomy or ureteroscopy), cost-effective phytotherapy options possessing antilithiatic activity, with minimal side effects as compared to allopathic treatments is being sought. The World Health Organization (WHO) reported that ~75% global population, mostly in the developing world, depends on botanical medicines for their basic healthcare needs with around 800 plants being used in indigenous system of medicines (Verma and Singh, 2008). Composite anti urolithiatic herbal formulations such as Cystone, Calcury Chandraprabhabati, Neeri and Uriflush have been developed by pharmaceutical companies for the treatment of kidney stones (Aggarwal,2014).

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The present study was aimed to assess the effect Terminalia arjuna against the oxalate caused cell damage and on the adhesion of COM crystals to cultured renal cells so as to establish a scientific basis for the anti-urolithiatic property of T. arjuna. T. arjuna belongs to the Combretaceae family and its bark extract is well known for treatment of cardiovascular ailments (Dwivedi and Chopra, 2014). It possesses many pharmacological properties including diuretic (Dwivedi, 2007), antioxidant (Shahriar et al., 2012) and antiatherogenic (Ram et al., 1997) as demonstrated in various studies. In a recent study, the inhibitory potential of T. arjuna was evaluated in vitro on CaOx crystallization and crystal growth (Chaudhary et al., 2010 and Mittal et al., 2015). Since, there is no permanent cure for urolithiasis, the long-term aim is to utilize these activities of T. arjuna and develop an adjunct therapy to prevent as well as cure kidney stones from recurring, without the burden of side effects associated with modern interventional procedures. Materials and methods

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2.1. Plant

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The dried bark of T. arjuna were purchased from Natural Remedies Pvt. Ltd., Bangalore, India. A voucher specimen (TRM 670) is available at the company. 2.2. Preparation of the AE of Terminalia arjuna

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The dried fine powdered T. arjuna bark was soaked in distilled water for 24 hours at 4°C. The extract was then filtered through muslin cloth followed by centrifugation at 10,000 rpm for 20 mins at 4°C and the filtrate was lyophilized to obtain the dried powder referred to as AE of T. arjuna bark. The dried AE was stored in labeled sterile bottles and kept at -20° C (Mittal et al., 2015).

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2.3. Preparation of AE for cell line studies

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For cell culture studies, a stock solution of the dried AE of T. arjuna was dissolved in dimethyl sulfoxide (DMSO) [final concentration of the DMSO in the highest concentration of plant extract tested did not exceed 0.4% (v/v) and did not affect the cell proliferation]. Further dilutions of the stock were done using serum free DMEM (Dulbecco’s Modified Eagles’s Media) and filtered by 0.22 µm syringe filter (Moriyama et al., 2007). 2.4. Cell culture Experimental studies were done using in vitro model of Normal rat epithelial derived renal tubular epithelial (NRK-52E) cell lines obtained from NCCS (National Centre for Cell Science), Pune, India. Cells were maintained as monolayers in DMEM. Media was supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin (100 units/mL)-Streptomycin (10,000 μg/mL). Cells were cultured in 25 cm2 tissue-culture treated flasks at 37ºC under 5% CO2 in humidified chambers (Aggarwal et al., 2010) for subsequent subculture in preparation for experiments. 2.5. Oxalate-induced cell injury

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A stock solution of 10 mM sodium oxalate was prepared and diluted to 2 mM in serum-free DMEM. NRK-52E cells were treated with 2 mM sodium oxalate in the absence and presence of different concentrations ranging from10-40 μg/mL of the AE for 48 hours (Jeong et al., 2005). Cystone drug at a concentration of 40 μg/mL was used as a positive control. 2.6. MTT Assay

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1x104 cells/well were seeded into a 96-well microplate and incubated at 37°C and 5% CO2 in humidified chambers to achieve 80% confluency. After incubation, cells were exposed to 2 mM sodium oxalate in the absence and presence of different concentrations (10, 20, 30 and 40 μg/mL) of AE and incubated for 48 hours at 37°C. At the end of the treatment, 25 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (final concentration of 0.5 mg/ml) was added to each well and incubated for 4 hours at 37°C. After incubation, supernatant was discarded and 200 μL DMSO was added to each well to solubilize the formazan product and kept at room temperature for 15-20 minutes. After gentle mixing, absorbance values were determined at a 570nm test wavelength and a 63nm reference wavelength to assess the cell viability using a microplate reader (Model 680, Bio-Rad) (Zhang et al., 2013).

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2.6. CaOx Crystal adhesion

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Cells were seeded on sterile glass coverslips placed in a 6-well plate at a density of 2x105 cells/coverslip and cultivated at 37°C and 5% CO2 in humidified chambers. At 80% confluency, cells were treated with 2 mm oxalate in the absence and presence of AE at a concentration of 40 μg/mL and incubated for 48 hours at 37°C. After the treatment the medium was removed and cells were washed twice with 1X PBS followed by fixation with 4% paraformaldehyde for 30 minutes. After incubation, cells were washed twice with 1X PBS and then observed under phase contrast and polarization upright microscope (BX53, Olympus Corporation, Japan) at a magnification of 20X to study cell-crystal interactions (Semangoen et al., 2008).

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2.7. Hoechst 33258 staining

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Cells were seeded on sterile glass coverslips placed in a 6-well plate at a density of 2x105 cells/coverslip and cultured at 37°C and 5% CO2 in humidified chambers to achieve 80% confluence. After incubation, cells were treated with 2 mM sodium oxalate in the absence and presence of. AE at a concentration of 40 μg/mL for 48 hours at 37°C. At the end of the treatment the medium was removed and cells were washed twice with 1X PBS followed by fixation with 4% paraformaldehyde for 30 minutes. After incubation, cells were washed twice with 1X PBS, stained with 5 μg/mL of Hoechst 33258 dye for 10 minutes at room temperature in the dark followed by washing twice with 1X PBS. Stained nuclei were observed under fluorescence upright microscope (BX53, Olympus Corporation, Japan) at a magnification of 20X (Allen et al., 2001).

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2.8. Annexin V/ Propidium Iodide staining

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6x105 cells were seeded into 60 mm dishes and incubated at 37°C and 5% CO2 in humidified chambers. At 80% confluency, cells were exposed to 2 mM sodium oxalate in the absence and presence of AE at a concentration of 40 μg/mL for 48 hours at 37°C. After the treatment, the subsequent procedure followed was in accordance to the instructions of BD PharminogenTM FITC ANNEXIN V Apoptosis Detection Kit 1 (catalogue no. 556547), where cell suspension and cells from monolayer were pooled together. The cells were washed with cold 1X PBS twice. The pellet was resuspended in 100 μL of 1X binding buffer followed by addition of 2 µL of FITC Annexin V and 2 µL of Propidium iodide (PI). The cells were gently vortexed and incubated for 15 minutes at room temperature in the dark. After incubation, 400 µL of 1X binding buffer was added to each group and then the cells were analyzed by flow cytometry (BD Accuri C6, BD Biosciences). 2.9. Detection of Active Caspase-3 6x105 cells were seeded into 60 mm dishes and incubated at 37°C and 5% CO 2 in humidified chambers. At 80% confluency, cells were treated with 2 mM sodium oxalate in the absence and presence of AE at a concentration of 40 μg/mL for 48 hours at 37°C. At the end of the treatment, the subsequent procedure followed was in accordance to the instructions of BDPharminogenTM FITC Active Caspase-3 Apoptosis Kit (catalogue no. 550480), where, cell suspension and cells from monolayer were pooled together. Cells were washed with cold PBS twice and resuspended in BD Cytofix/Cytoperm solution. After incubation on ice for 20 minutes, BD Cytofix/Cytoperm solution was discarded. The cells were washed twice with 1X BD Perm/Wash buffer at room temperature. The

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cells were then resuspended in the 1X BD Perm/Wash buffer plus 10 µL of antibody and incubated for 30 minutes at room temperature. After incubation, the cells were washed with 0.5 mL of 1X BD Perm/Wash buffer and then resuspended in 0.5 mL of 1X BD Perm/Wash buffer for analysis by flow cytometry (BD Accuri C6, BD Biosciences). 2.10. Statistical Analysis

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Results

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3.1. Effect of aqueous extract on oxalate injured renal epithelial cells (MTT Assay)

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Statistical procedures were performed with GraphPad Prism software version 6.01. The statistically different groups were identified by one-way analysis of variance (ANOVA), followed by Dunnet’s multiple comparison tests. Results were expressed as the mean ± SD. A p-value of <0.05 was considered significant. All the experiments were performed three times, each time in triplicate.

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The cytoprotective potential of AE of Terminalia arjuna against oxalate induced injury to NRK-52E cells was assessed after 48 hours of treatment (Fig. 1). Cells treated with serum free defined medium were considered as the untreated control group. On exposing the renal epithelial cells to the solvent alone (0.4% DMSO) or AE (40 μg/mL containing 0.4% DMSO), no adverse effect on the cells was observed. which was in contrast to when cells were exposed to 2 mM oxalate wherein, there was a sharp plunge in viability from 100% in untreated cells to 23.75 ± 1.66% (p<0.001). When the oxalate injured cells were co-treated for 48 hours with AE, the viability significantly increased in a concentration dependent manner. The percentage viability with 10 μg/mL, 20 μg/mL, 30 μg/mL and 40 μg/mL of extract was 25.89 ± 1.44%, 33.22 ± 1.57%, 39.71 ± 1.37% and 48.47 ± 1.83%, respectively. Cystone at a concentration of 40 μg/mL was used as a positive control and also showed cytoprotection from oxalate induced injury. 3.2. Disruption of CaOx crystal-cell interaction by aqueous extract

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The interaction of CaOx crystals with NRK-52E cells was studied to demonstrate the oxalate induced injuries and crystal adhesion on the surface of renal epithelial cells in the absence and presence of AE for 48 hours (Fig. 2). Cells incubated with serum free medium were taken as untreated cells i.e. control group (Fig. 2A). The cells treated with the solvent system (Fig. 2B) and AE (Fig. 2C) showed healthy cellular morphology as shown by the yellow arrows, indicating that there was no adverse effect to the cells. After incubation of the NRK-52E cells with 2 mM oxalate, a noticeable change in cell morphology was manifested by a decrease in size and increase in the granularity (red arrows). In addition, CaOx crystals attached to cell surface of renal epithelial cells were observed and these crystals remained adhered to the cells even after several washes using PBS (Fig. 2D). These adhered crystals caused cell damage and subsequent cell death which was apparent by fall in the number of viable cells w.r.t control group. The addition of 40 μg/mL of AE to oxalate injured NRK-52E disrupted the interaction between cells and CaOx crystals, showing more viable cells, fewer crystals and loss of crystal adherence to cells, which could be ascertained from polarization images (Fig. 2E). These cells appeared to be akin to untreated cells when observed under the microscope (blue arrows). The treatment with Cystone also showed similar protective effects which led to more viable cells and loosely bound crystals (Fig. 2F). 3.3. Detection of Oxalate induced apoptosis by Hoechst staining

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The morphological changes in cell nuclei as a consequence of oxalate exposure to renal cells were determined by staining NRK-52E cells with Hoechst 33258 dye (Fig. 3). The untreated NRK-52E cells appeared to be in healthy morphology with intact cell membrane and chromatin and were weakly stained (Fig. 3A). The cells treated with the solvent system (Fig. 3B) and AE (Fig. 3C) also exhibited healthy cellular morphology and were lightly stained, indicating that there was no harmful effect to the cells. However, when NRK-52E cells were incubated with oxalate (Fig. 3D), a substantial level of cell death w.r.t. untreated cells was observed. The cells showed marked changes in cell morphology with condensed and fragmented chromatin and were brightly stained indicating early signs of apoptosis. The effect of 40 μg/mL of AE on oxalate treated NRK-52E cells was assessed and it was evident that the treatment protected the cells from oxalate induced apoptosis as more viable cells with intact cellular membrane and chromatin were observed (Fig. 3E) w.r.t. the oxalate injured cells. The addition of Cystone also reduced oxalate induced apoptosis and protected the renal cells which led to an increase in viable cell number (Fig. 3F) w.r.t oxalate injured cells.

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3.4. Diminution of apoptotic cell death by aqueous extract

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Oxalate-induced apoptosis was evaluated in NRK-52E cells after 48 hours of exposure to oxalate by staining with Annexin V and PI (Fig. 4). There was no substantial apoptosis in the untreated cells as majority of cells were in lower left quadrant depicting viable cell percentage. Treatment of cells with the solvent system and AE did not lead to any significant alteration in cell viability. When renal cells were exposed to oxalate for prolonged duration, the time-course cell death assay showed that percent of cells undergoing early apoptosis (lower right quadrant) gradually increased from 0.3% in control to 50% in oxalate treated. Moreover, the percent of late apoptotic cells (top right quadrant) also increased to 13.2% w.r.t control. The co-treatment with 40 μg/mL of AE on oxalate injured cells was evaluated and it was seen that the percentage of early apoptotic and late apoptotic cells was significantly reduced to 37.4% and 7.9% respectively, signifying that AE protected the renal cells from oxalateinduced apoptosis. The treatment of oxalate treated renal cells with Cystone also improved cell viability to 47.2% compared to oxalate injured cells.

Discussion

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Oxalate-induced apoptosis in NRK-52E cells was further assessed by Anti-Active Caspase-3 antibody, staining the cells undergoing apoptosis (Fig. 5). Treatment of cells with the solvent system and AE caused no harmful effects to cells. After incubation of renal cells with 2 mM oxalate for 48 hours, the percent of cells undergoing apoptosis (depicted by M2) was gradually increased from 22.3% in control to 72.9% in oxalate treated cells. The effect of 40 μg/mL of AE on oxalate treated cells was evaluated, and it was observed that the percent of apoptotic cells was significantly decreased to 58.7%, indicating that AE reduced oxalate-induced apoptosis. The Cystone treatment on oxalate injured renal cells also reduced the percentage of apoptosis to 61.1% though it was less efficacious in comparison to oxalate damaged cells treated with AE.

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In the present study, we demonstrated the cytoprotective effect of T. arjuna against oxalate induced damage and cell death either by apoptosis or necrosis. Numerous studies have been conducted confirming oxalate and/or CaOx crystal toxicity to renal epithelial cells, which results in a redistribution of phosphatidylserine on the damaged cell surface (Wiessner et al., 1999), increased reactive oxygen species (ROS) production (Scheid et al., 1996), enhanced expression of immediate early genes (Lieske et al., 1992) and cell death by both apoptosis and necrosis (Miller et al., 2000). Oxalate, being the metabolic end product, excreted in the urine by kidney (Knight et al., 1979 and Koul et al., 1994a), is a key component responsible for the formation of CaOx stones at pathophysiological concentrations (Menon and Koul, 1992). Under normal circumstances, non-stone formers produce these crystals and excrete them into urine with urinary oxalate concentration of 0.22 mM. Under mild hyperoxaluric condition, the oxalate concentration is increased to 0.45 mM and can go as high as 1.5 mM during primary hyperoxaluria (Koul et al., 1994b and Kohjimoto et al., 1999). These crystals transform into a stone when they are occluded in the kidneys of stone formers. Ergo, interplay of CaOx crystals with renal epithelial cells play the most crucial role for the development of kidney stones.

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To create hyperoxaluric conditions akin to the in vivo kidney environment, NRK-52E cells (an immortal cell line derived from normal rat kidney) were exposed to 2 mM oxalate for 48 hours. The authors chose Cystone, an herbal formulation manufactured by The Himalaya Drug Co., as a positive control for our studies. Cystone is a composite herbal formulation comprising of a number of medicinal plants and herbal extracts which possess antilithiatic, lithotriptic and diuretic properties, which averts supersaturation of stone forming agents in urine thereby, inhibiting the crystallization process and kidney stone formation. It has also been seen to dissolve the calculus by dissolving the mucin, which binds insoluble stone particles together (Mago et al., 1989 and Kumaran and Patki, 2011). However, as the therapeutic actions of poly herbal formulations(PHF) are thought to be the synergistic effects of all the components, our aim was to find a comparable single plant formulation. The use of a single plant formulation could eliminate some of the expensive quality control issues related to the manufacturing of PHF which require standardization of the raw components whose chemical components may be affected by the area from where they are procured, as well as the manner in which they are harvested and processed (Bauer and Tittel, 1996). This study was undertaken to explore a single plant based formulation for the effective management of kidney stones. In the in vitro cell culture study, the aqueous extract of T. arjuna protected the NRK-52E cells and reduced the oxalate induced damage to cells in a dose-dependent manner as verified by MTT assay, which reflects the metabolism of viable cells. The adhesion of CaOx crystals to the cell surface of NRK-52E was also studied in the absence and presence of AE. It was observed that when these cells were exposed to oxalate, CaOx crystals adhered

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tightly to renal cells despite several PBS washings. These crystals caused cell damage and death which was evident by reduction in cell density as compared to untreated cells. It could be postulated that the AE reduced the oxalate induced injury to the renal cells by disrupting the interaction of CaOx crystals with the cells which was evident by increase in the number of viable cells and loosely bound crystals. Since, aqueous extract contains both primary and secondary metabolites, it could be possible that these metabolites either coated the crystals or interacted with the receptors exposed on the outer leaflet of the membrane as a consequence of oxalate exposure or crystal binding molecules, thus attenuating the CaOx crystallization and attachment of these crystals to the cells.

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We also demonstrated that exposure to oxalate induced apoptosis leading to increased cell death. The morphological changes in cell nuclei on exposure to oxalate were visualized by staining cells with Hoechst 33258 dye. The cells treated with oxalate showed marked changes in cellular morphology with brightly stained condensed and fragmented chromatin, which is a key event signifying apoptosis, as compared to weakly stained control group. After the co-treatment with AE, cells appeared morphologically similar to untreated cells which was apparent by the larger number of lightly stained viable cells with intact cellular membrane and fewer apoptotic bodies, showing reduced level of apoptosis. This was further confirmed and quantified by Annexin V/PI and AntiActive Caspase-3 antibody staining. When renal cells were injured with oxalate, the number of cells that bind to Annexin V (early apoptosis, lower right quadrant) increased, indicating that phosphatidylserine was exposed on the cell luminal surface upon oxalate treatment since Annexin V has affinity for anionic phospholipid (Cao et al., 2001). This observation is consistent with literature demonstrating that exposure to high concentration of oxalate results in the relocation of phosphatidylserine to the cell surface (Wiessner et al., 1999) and the ability of phosphatidylserine to act as a site for crystal attachment (Bigelow et al., 1996). We observed that treatment with AE significantly reduced the number of apoptotic cells by disrupting the interaction of CaOx crystals with the cells, thereby rendering protection.

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In our study, we observed that majority of cell death upon exposure to oxalate was due to apoptosis. This is in keeping with reports which state that cellular exposure to high oxalate load in vitro (Hackett et al., 1994) and in vivo (Khan et al., 1992) elicits apoptosis in renal epithelial cells, leading to cell death by a process involving increased oxidant stress (Scheid et al., 1996). These ROS which are generated dissipate the mitochondrial membrane potential followed by an increased mitochondrial permeability and release of pro-apoptotic factors. These factors in turn form an apoptotic complex and trigger the activation of cellular caspases, which are markers of cells undergoing apoptosis (Petronilli et al., 2001). Caspase-3 is a key protease that is activated during the early stages of apoptosis. Our results confirmed that when renal cells were exposed to oxalate, the number of apoptotic cells significantly increased (shown by the marked margin of M2) which were detected by anti-active caspase-3 antibody. The addition of AE reduced the number of apoptotic cells thus proving its ability to protect against oxalate-induced cell oxidative stress which is a trigger for apoptotic cell death. Conclusions

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The results of this study lend scientific evidence for the antilithiatic ability of the aqueous extract of T. arjuna. The basis of the antilithiatic effect may lie in the active biomolecules present in AE of T.arjuna which have been shown to inhibit the binding of the CaOx crystals to the renal epithelial surface and/or interaction of oxalate ions with calcium ions. Interference with various processes associated with the formation of stones such as CaOx crystallization and retention to renal cells, therefore, seems a possible therapeutic strategy for the prevention of recurrent stone disease. Based on this study the efficacy of a single plant herbal formulation such as T. arjuna towards the oxalate-induced cell injury could be recommended.

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Funding: This work was supported by the Department of Science and Technology (DST), Government of India, New Delhi, India [SR/SO/HS/132/2010]. Conflict of interest: The authors declare that they have no conflict of interest. Acknowledgement: This study (SR/SO/HS/132/2010) was financially supported by the Department of Science and Technology (DST), Government of India, New Delhi, India. We would like to thank the Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, India for providing the necessary facilities to carry out this study.

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Fig.1 Efficacy of aqueous extract against oxalate damage in NRK-52E cells Effect AE of T. arjuna on oxalate injured NRK-52E cell viability assessed by MTT assay. Data are mean ± S.D of three independent observations. ns: not significant. *** p < 0.005, **** p < 0.0001 versus untreated control; and # p < 0.05, ## p < 0.005, ### p < 0.001 versus oxalate injury control

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Fig.2 Inhibition of CaOx crystal adhesion to renal cells by aqueous extract Effect of AE of T. arjuna on CaOx crystal adherence in oxalate induced injury to NRK-52E cells, visualized under polarization and phase contrast at magnification 20X and scale bar 100 microns

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Fig.3 Detection of oxalate induced apoptosis by Hoechst staining Effect of AE of T. arjuna on induction of apoptosis in oxalate induced injury to NRK-52E cells, visualized under fluorescence microscopy at magnification 20X and scale bar 100 microns. The inset images are zoomed in areas highlighted by the white arrows

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Fig.4 Dimunition of apoptotic cell death by aqueous extract Flow cytometry analysis showing the effect of AE of T. arjuna on induction of apoptosis in oxalate induced injury to NRK-52E cells, visualized by Annexin V/PI staining

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Fig.5 Reduction of pro-apoptotic Caspase 3 in oxalate injured cells Flow cytometry analysis showing the effect of AE of T. arjuna on induction of apoptosis in oxalate induced injury to NRK52E cells, visualized by Anti-Active Caspase-3 antibody staining

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